JP2006501806A - Optineurin and glaucoma - Google Patents

Abstract

There is an increased risk of optineurin-related glaucoma or optineurin-related glaucoma testing the sample for the presence of glaucoma or specific mutations associated with an increased risk of glaucoma in the gene optineurin Or a method for diagnosing its absence is described. Also described are methods of testing a sample for the presence of denaturation in expression of the optineurin polypeptide, and methods of testing the sample for the presence of alteration of the activity of the optineurin polypeptide. A method for treating glaucoma using an optineurin therapeutic agent is also described.

Description

RELATED APPLICATION This application is a U.S. application filed on January 30, 2002, which claims the benefit of US Provisional Application No. 60 / 344,754, filed December 24, 2001, which is incorporated herein by reference in its entirety. This is a continuation application of US 10 / 281,457, filed on October 25, 2002, which is a continuation application of US 10 / 090,118, filed on February 28, 2002, which is a continuation-in-part of 10 / 060,981.

Government Support This invention was supported in whole or in part by a grant RO1-EY09947 from the National Institutes of Health (National Eye Research Institute). The US government has some rights in this invention.

  Glaucoma is a progressive optic neuropathy characterized by a specific pattern of visual field loss and optic disc damage resulting from several different diseases affecting the eye. About 2.47 million people in the United States suffer from glaucoma (Quigley, HA and Vitale, S., Invest. Ophthalmol. Vis. Sci., 38:83, (1997)), and more than 100,000 Americans annually The condition is expected to develop. Furthermore, it is estimated that 67 million people worldwide have glaucoma (Quigley, H.A., Br. J. Ophthalmol., 80: 389, (1996)). The most common type of this condition is primary open angle glaucoma (POAG). Glaucoma optic nerve damage and characteristic visual field loss are two major clinical signs of this condition (Crick, RP, Lancet, 1: 205, (1974); Quigley, HA, N. Engl. J Med. 328: 1097, (1993); Wilson, R. and Matrone, J., `` The Glaucomas, '' Volume 2, pages 753-768 (Ritch, SM and Krupin, T., St. Louis: Mosby, 1996)) . The most common known risk factor for glaucomatous damage is elevated intraocular pressure (IOP), which does not correspond to the disease itself, and a number of other risk factors are currently being investigated. Yes. About 1/3 to 1/2 of patients with POAG (i.e. 1.2 million in the United States alone) are within the statistically normal range with an IOP less than 22 mmHg (Tielsch, JM et al., JAMA, 266: 269, (1991); Hitchings, RA, Br. J. Ophthalmol., 76: 494, (1992); Grosskreutz, C. and Netland, PA, Int. Ophthalmol. Clin., 34: 173, (1994) Werner, EB, “The Glaucomas,” Volume 2, pages 768-797 (Ritch, SM and Krupin, T., St. Louis: Mosby, 1996.) These patients have low-tension glaucoma or normal-tension glaucoma. (LTG or NTG) and is typical of optic disc glaucomatous depression and visual field loss (Hitchings, RA and Anderton, SA, Br. J. Ophthalmol., 67: 818, (1983)).

During the past decade, eight different loci have been identified for various glaucoma genotypes. Two loci have been reported for primary congenital glaucoma (PCG), one for juvenile-onset glaucoma (JOAG), and five for adult-onset POAG (Sarfarazi, M. and Stoilov, I., `` Ophthalmic Fundamentals: Glaucoma (Sassani, JW (Slack Inc., Thorofare, NJ, 1999), pages 15-31), but the causative gene has two rare forms of this condition: PCG (Stoilov, I. et al., Hum Mol. Genet., 6: 641, (1997)) and JOAG (Stone, EM et al., Science, 275: 668, (1997)). Ongoing studies show that cytochrome P4501B1 is the major PCG Genes (i.e. 85% of familial cases and 33% of sporadic cases) have been shown (Stoilov, I. et al., Am. J Hum. Genet., 62: 573, (1998) ), A small subset of both JOAG and POAG subjects (ie 3.0-4.0%), mainly involving mutations in the myosillin gene, most of which are the same in JOAG cases. There are other JOAG families that have been defined (ie 2.0-2.5%) but this gene is not mutated (Stoilova, D. et al., J. Med. Genet., 35: 989, (1998)). In addition, only a small number of mutations have been reported in adult-onset POAG cases (ie 1.0-1.5%), and no other genes have been identified so far that are responsible for the adult-onset POAG phenotype.
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  As described herein, mutations in one gene, optineurin, have been linked to increased risk of glaucoma and glaucoma. Accordingly, the present invention relates to a method of diagnosing the presence or absence of an increased risk of optineurin-related glaucoma or optineurin-related glaucoma in an individual.

  The method includes detecting the presence or absence of a degeneration (eg, a mutation) in the optineurin gene, or detecting an alteration in expression or composition of the optineurin polypeptide. This denaturation can be quantitative, qualitative, or both quantitative and qualitative. The method can also include detecting the presence or absence of denaturation in the activity of the optineurin polypeptide. The presence of degeneration in a gene, or the presence of a degeneration in the expression or composition of an optineurin polypeptide, or the presence of a degeneration in the activity of an optineurin polypeptide is related to optineurin-related glaucoma or optineurin-related. It is an indicator of an increased risk of glaucoma. The absence of degeneration in a gene, or the absence of degeneration in the expression or composition of an optineurin polypeptide, or the absence of degeneration in the activity of an optineurin polypeptide is related to optineurin-related glaucoma or optineurin-related. It is an indicator that there is no increased risk of glaucoma.

  The invention further provides a nucleic acid encoding an optineurin or an optineurin interacting polypeptide; an optineurin polypeptide or an optineurin interacting polypeptide; an agent that alters the activity of an optineurin polypeptide or an optineurin interacting polypeptide To an increased risk of glaucoma or glaucoma by administering an optineurin therapeutic agent such as Methods for treating glaucoma or an increased risk of glaucoma also include the use of antisense therapy, ribozymes, and homologous recombination. The present invention further relates to the use of an optineurin therapeutic for the manufacture of a medicament for the treatment of glaucoma or the treatment of increased risk of glaucoma.

  The method of the present invention makes it possible to screen for this disease in individuals at high risk, such as the elderly, and to facilitate both therapeutic and prophylactic treatment of glaucoma.

  The foregoing and other objects, features and advantages of the present invention will become apparent from the following more specific description of preferred embodiments of the invention, as illustrated in the accompanying drawings.

The present invention relates to genes identified as being associated with adult-onset primary open angle glaucoma (POAG). As described herein, Applicants have identified a series of mutations in one gene, optineurin. This mutation is a major cause of the adult-onset low-tension glaucoma (LTG) / primary open-angle glaucoma (POAG) phenotype within each family. Applicants have further identified mutations in the optineurin gene that are associated with an increased risk of glaucoma. Optineurin was first identified as a tumor necrosis factor-α (TNF-α) inducible protein (Li, Y. et al., Mol. Cell. Biol., 18: 1601, (1998)), FIP-2 (adenovirus E3-15.7K interacting protein 2)). This is followed by huntingtin interacting protein L (HYPL) (Faber, PW et al., Hum. Mol. Genet., 7: 1463, (1998)), NEMO-related protein (NRP) (Schwamborn, K. et al., J Biol. Chem., 275: 22780, (2000)), transcription factor IIIA interacting protein (TFIIIA-INTP) (Moreland, RJ et al., Nucleic Acids Res., 28: 1986, (2000)), and RAB8 interaction. It was also identified as a protein (Hattula, K. and Peranen, J., Curr. Bio., 10: 1603, (2000)). In this study, the use of as the new name of this protein "optineurin (optineurin)" (optic nerve disease-induced (Opt ic Neur opathy In ducing) protein). The presence of one or more mutations in optineurin may interfere with the DNA or protein binding ability of optineurin, leading to premature cleavage of the protein by another mutation. Evidence indicates that direct interaction between optineurin and E3-14.7K protein mediates apoptosis, inflammation or vasoconstriction, possibly utilizing the TNF-α or Fas-ligand pathway. Optineurin also functions through interactions with other proteins in cell morphogenesis and membrane transport (RAB8), vesicle transport (huntingtin), transcriptional activation (TFIIIA) and assembly or activity of two kinases .

  Accordingly, the present invention relates to a method of treating glaucoma, and detecting the presence or absence of denaturation in an optineurin gene or an optineurin polypeptide, or altering the activity of an optineurin polypeptide or optineurin. It also relates to methods and kits for detecting the presence or absence of an increased risk of glaucoma or glaucoma in an individual by detecting denaturation of interacting polypeptides. Glaucoma associated with the presence of one or more degenerations in the optineurin gene or optineurin polypeptide is referred to herein as “optineurin-related glaucoma” and is referred to as an optineurin gene or an optineurin polypeptide. The increased risk of glaucoma associated with one or more degenerations therein is referred to herein as “increased risk of optineurin-related glaucoma”.

  As used herein, the term `` glaucoma '' refers to hereditary glaucoma such as primary congenital glaucoma or childhood glaucoma; primary open-angle glaucoma, including both early-onset glaucoma and adult or late-onset POAG ( POAG); secondary glaucoma; pigmentary glaucoma; low-tension glaucoma (LTG); and normal-tension glaucoma (NTG). In certain embodiments, the glaucoma can be low-tension glaucoma (LTG), normal-tension glaucoma (NTG), or primary open-angle glaucoma (POAG). As used herein, “increased risk” of glaucoma refers to the likelihood that an individual will develop glaucoma, which is statistically more significant than the likelihood that another individual or population will develop glaucoma. .

Diagnostic Methods and Kits for Diagnosis Methods Based on Optineurin Genes and Nucleic Acids In one embodiment of the invention, diagnosis of optineurin-related glaucoma, or diagnosis of increased risk of optineurin-related glaucoma comprises glaucoma or glaucoma. This is done by detecting the presence or absence of denaturation in the optineurin gene that is associated with an increased risk. As used herein, the term `` optineurin gene '' refers to an optineurin polypeptide (tumor necrosis factor-α (TNF-α) inducible protein (FIP-2); huntingtin interacting protein L (HYPL)); Refers to nucleic acid (eg, DNA, RNA, cDNA) encoding NEMO-related protein (NRP); transcription factor IIIA interacting protein (TFIIIA-INTP); also known as RAB8 interacting protein. As used herein, “gene” includes not only translated nucleic acids but also non-translated nucleic acids (eg, promoter regions).

  Sequence information includes GenBank accession number AF420371-3; SEQ ID NOs: 1, 3, and 5 (nucleic acids encoding optineurin isoforms 1, 2, and 3, respectively); and SEQ ID NOs: 2, 4, and 6 (respectively See optineurin isoforms 1, 2 and 3). SEQ ID NO: 1 includes an optineurin comprising a 5 ′ untranslated region from nucleotides 1-310, a coded isoform from nucleotides 311 to 2044 (SEQ ID NO: 2), and a 3 ′ untranslated region from nucleotides 2045-2077. Codes isoform 1. SEQ ID NO: 3 comprises an optineurin comprising a 5 ′ untranslated region from nucleotides 1 to 89, an encoded isoform from nucleotides 90 to 1823 (SEQ ID NO: 4), and a 3 ′ untranslated region from nucleotides 1824 to 1856. Codes isoform 2. SEQ ID NO: 5 is a sequence of optineurin, including the 5 ′ untranslated region from nucleotides 1-241, the encoded isoform from nucleotides 242-275 (SEQ ID NO: 6), and the 3 ′ untranslated region from nucleotides 1976-2008. Codes isoform 3.

  See also, for example, AH009711; AF061034; AF283519-27. Also, Li, Y. et al., Mol. Cell. Biol., 18: 1601, (1998); Faber, PW et al., Hum. Mol. Genet., 7: 1463 (1998); Schwamborn, K. et al., J Biol Chem., 275: 22780, (2000); Moreland, RJ et al., Nucleic Acids Res., 28: 1986, (2000); and Hattula, K. and Peranen, J., Curr. Bio., 10: 1603, See also (2000). The entire teachings of these GenBank accession numbers, particularly GenBank accession number AF420371-3, and the entire teachings of these references are incorporated herein by reference in their entirety.

  A `` denaturation '' is a change in a nucleic acid encoding an optineurin polypeptide (e.g., insertion, deletion, or deletion of one or more nucleotides) compared to a known sequence of the nucleic acid encoding optineurin. Change). This denaturation results in a frameshift mutation, a mutation in the optineurin gene, such as an insertion or deletion of a single nucleotide or multiple nucleotides; a change in at least one nucleotide resulting in a change in the encoded amino acid; an immature stop codon At least one nucleotide change that results in the occurrence of; a deletion of some nucleotides that results in the deletion of one or more amino acids encoded by the nucleotide; an inequality that results in an interruption in the coding sequence of the gene Insertion of one or several nucleotides, such as by replacement or gene conversion; replication of all or part of a gene; transfer of all or part of a gene; or rearrangement of all or part of a gene. There may be multiple such mutations in a single gene. Such a sequence change causes a mutation of the polypeptide encoded by the optineurin gene. For example, if the mutation is a frameshift mutation, it may result in a change in the amino acid encoded by the frameshift and / or may result in the generation of an immature stop codon, resulting in cleavage. Production of the resulting polypeptide. Alternatively, a degeneration associated with glaucoma can be a synonymous mutation in one or more nucleotides (ie, a mutation that does not cause a change in the polypeptide encoded by the optineurin gene). Such denaturation may modify the splicing site, affect mRNA stability or transport, or otherwise affect gene transcription or translation. An optineurin gene having any of the mutations described above is referred to herein as a “mutant gene”.

  In certain embodiments of the invention, the alteration is a GAG to AAG change at codon 50 of the optineurin gene; an AG insertion after codon 127; or a CGG to CAG change at codon 545. These degenerations are associated with glaucoma, and glaucoma is diagnosed by the presence of one or more of these degenerations. In another specific embodiment, the degeneration is a change from ATG to AAG at codon 98, the presence of this degeneration being an indicator of an increased risk of glaucoma, and the presence of it is indicative of the risk of glaucoma. An increase is diagnosed.

  The first method of diagnosing glaucoma or an increased risk of glaucoma uses hybridization methods such as Southern analysis (`` Current Protocols in Molecular Biology '', Ausubel, F. et al., John Wiley & Sons (2001 (Including the supplement to); this document is incorporated herein by reference in its entirety). For example, a genomic DNA, RNA, or cDNA test sample may be, for example, an individual suffering from glaucoma, having a glaucoma defect, or suspected of having an increased risk of glaucoma. Obtained from an individual ("test individual"). The individual can be an adult, a child, or a fetus. Test samples are blood samples, serum samples, lymph fluid samples, fluid samples from the eye (e.g., anterior chamber fluid), amniotic fluid samples, cerebrospinal fluid samples, or skin, muscle, buccal mucosa, conjunctival mucosa, placenta , From any source containing nucleic acids (eg, DNA, RNA), such as tissue samples from the gastrointestinal tract or other organs. A test sample of DNA derived from fetal cells or tissues can be obtained by a suitable method such as by amniocentesis or serous fur collection. A DNA, RNA, or cDNA sample is then examined to determine if there is any denaturation in the optineurin gene.

  If desired, sample amplification (eg, by polymerase chain reaction) can be performed prior to assessing the presence or absence of denaturation in the optineurin gene. Amplification can be used on all or part of the nucleic acid containing the optineurin gene, where this part is part of the optineurin gene that contains the denaturation (e.g., exon 4, exon containing the denaturation, as described below). 6 or other exons, such as one or more exons). In a preferred embodiment, this portion comprises at least one exon of the optineurin gene.

  The presence or absence of denaturation can be indicated by hybridization of a gene in genomic DNA, RNA, or cDNA to a nucleic acid probe. As used herein, a “nucleic acid probe” is a single-stranded oligonucleotide that hybridizes to a target gene (optineurin). The appropriate length of the probe is typically in the range of 15 to 30 nucleotides. Short probes generally require lower temperatures to form a sufficiently stable hybrid complex with the template. The nucleic acid probe can be a DNA probe or an RNA probe, and the nucleic acid probe comprises at least one denaturation in the optineurin gene. Probes can include whole genes, gene fragments, vectors containing genes, exons of genes, and the like.

  In order to diagnose the presence of glaucoma or an increased risk of glaucoma, a hybridization sample is formed by contacting a test sample containing the optineurin gene with at least one nucleic acid probe. The hybridization sample is maintained under conditions sufficient to allow specific hybridization of the nucleic acid probe to the optineurin gene. As used herein, “specific hybridization” refers to exact hybridization (eg, no mismatch). Specific hybridization can be performed, for example, under high stringency conditions or moderate stringency conditions.

  “Stringency conditions” for hybridization are terminology in the art that refers to incubation and wash conditions, eg, temperature and buffer concentration conditions that allow for hybridization of a particular nucleic acid to a second nucleic acid. is there. The first nucleic acid can be completely (i.e., 100%) complementary to the second nucleic acid, or the first and second nucleic acids can share some degree of complementarity (e.g., 70%, 75%, 85%, 95%, 98%). For example, certain high stringency conditions can be used to distinguish nucleic acids that are completely complementary from those that are less complementary.

  “High stringency conditions”, “moderate stringency conditions” and “low stringency conditions” for nucleic acid hybridization are described in “Current Protocols in Molecular Biology”, pages 2.10.1-2.10.16 and 6.3.1-6. (Ausubel, FM et al., “Current Protocols in Molecular Biology”, John Wiley & Sons, (1998)), the teachings of which are incorporated herein by reference. The exact conditions that determine the stringency of hybridization are the ionic strength of destabilizing agents such as formamide and denaturing agents such as SDS (e.g. 0.2 x SSC, 0.1 x SSC), temperature (e.g. room temperature, 42 ° C, 68 ° C). ) And concentration, as well as factors such as the length of the nucleic acid sequence, base composition, the proportion of mismatches between hybridizing sequences, and the frequency of occurrence of a subset of that sequence within other non-identical sequences. Accordingly, conditions of high, moderate or low stringency can be determined empirically.

  A given sequence hybridizes (for example, selectively) to the most similar sequence in a sample by changing the hybridization conditions from a stringency level at which no hybridization occurs to a level at which hybridization is first observed. The conditions that make it possible can be determined.

Exemplary conditions describe the determination of washing conditions at moderate or low stringency conditions, Krause, MH and SA Aaronson, Methods in Enzymology, 200: 546-556, (1991). Also, in, Ausubel Others are described in “Current Protocols in Molecular Biology”, John Wiley & Sons, (1998). Washing is usually a step where conditions are set to determine the minimum complementarity level of the hybrid. In general, starting from the lowest temperature at which only homologous hybridization occurs, each time the final wash temperature is reduced by 1 ° C (while keeping the SSC concentration constant), the maximum degree of mismatch between hybridizing sequences is 1%. To increase. In general, doubling the concentration of SSC increases _Tm by -17 ° C. Using these guidelines, the wash temperature can be empirically determined for high, medium or low stringency depending on the level of mismatch to be obtained.

  For example, a low stringency wash can include a 10 minute wash at room temperature in a solution containing 0.2 × SSC / 0.1% SDS, while a moderate stringency wash is 0.2 × SSC / 0.1%. Washing at 42 ° C for 15 minutes in a pre-warmed solution containing 42% SDS (42 ° C) can be included, and high stringency washing can be performed in advance with 0.1x SSC / 0.1% SDS. Washing at 68 ° C. for 15 minutes in warm (68 ° C.) solution can be included. Furthermore, the washing can be performed repeatedly or continuously as is known in the art to obtain the desired result. By changing one or more parameters given by way of example as is known in the art, while maintaining the same degree of identity or similarity between the target nucleic acid molecule and the primer or probe used. Equivalent conditions can be determined.

  In particularly preferred embodiments, the hybridization conditions for specific hybridization are high stringency. Thereafter, standard methods are used to detect specific hybridization, if present. If specific hybridization occurs between the nucleic acid probe and the optineurin gene in the test sample, the optineurin gene has the denaturation present in the nucleic acid probe. In this method, a plurality of nucleic acid probes can be used simultaneously. The specific hybridization of any one of the nucleic acid probes is an indicator of the presence of degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma, and thus is associated with optineurin An increased risk of glaucoma or optineurin-related glaucoma is diagnosed. The absence of specific hybridization is an indicator of the absence of degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma, and thus there is no optineurin-related glaucoma Or an increased risk of optineurin-related glaucoma is diagnosed.

  In another hybridization method, Northern analysis (Current Protocols in Molecular Biology, Ausubel, F. et al.) Is used to identify the presence or absence of degeneration associated with glaucoma or an increased risk of glaucoma. John Wiley & Sons (see above)). For Northern analysis, an RNA test sample is obtained from an individual by appropriate means. As mentioned above, specific hybridization of nucleic acid probes to RNA from an individual is an indication that there is degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma, and thus This diagnoses an increased risk of optineurin-related glaucoma or optineurin-related glaucoma. As noted above, the absence of specific hybridization of nucleic acid probes to RNA from an individual is an indication of the absence of degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma. Thus, it is diagnosed that there is no increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

  For representative examples of the use of nucleic acid probes, see, eg, US Pat. Nos. 5,288,611 and 4,851,330.

  Alternatively, peptide nucleic acid (PNA) probes can be used in place of nucleic acid probes in the hybridization methods described above. PNA is an organic base (A, G, C, T or U) that has a peptide-like inorganic main chain such as an N- (2-aminoethyl) glycine unit and is attached to the nitrogen of glycine by a methylenecarbonyl linker. (See, for example, Nielsen, PE et al., Bioconjugate Chemistry, 1994, 5, American Chemical Society, page 1, (1994). PNA probes are specific for genes with degeneration associated with glaucoma. As noted above, the specific hybridization of PNA probes to RNA from individuals can be found in the optineurin gene associated with increased risk of glaucoma or glaucoma. It is an indicator of the presence of degeneration, and thus it diagnoses an increased risk of optineurin-related glaucoma or optineurin-related glaucoma. The absence of specific hybridization of the PNA probe to RNA from an individual is an indication of the absence of degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma, and thus This diagnoses that there is no increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

  In another method of the present invention, mutation analysis by restriction digestion can be used to detect a mutated gene or a gene containing a degeneration if mutation or denaturation in the gene results in the creation or elimination of a restriction site. A test sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify the optineurin gene (and its flanking sequence if necessary) in the genomic DNA of the test sample from the test individual. RFLP analysis is performed as described (see Current Protocols in Molecular Biology (above)). The digestion pattern of the relevant DNA fragment indicates the presence or absence of mutations or degenerations in the optineurin gene that are associated with glaucoma or an increased risk of glaucoma, thus The presence or absence of an increased risk of glaucoma-related optineurin is diagnosed.

  Sequence analysis can also be used to detect specific alterations in the optineurin gene. A test sample of DNA or RNA is obtained from the test individual. PCR or other suitable method can be used to amplify the gene and / or its flanking sequences if necessary. Standard methods are used to determine the sequence of an optineurin gene or a fragment of the gene (eg, one or more exons), or a cDNA or cDNA fragment, or an mRNA or mRNA fragment. The sequence of the gene, gene fragment, cDNA, cDNA fragment, mRNA, or mRNA fragment is suitably compared with the known nucleic acid sequence of the gene, cDNA, or mRNA, as appropriate. The presence of degeneration in the optineurin gene indicates that the individual has a glaucoma or a degeneration associated with an increased risk of glaucoma, and thus this may lead to optineurin-related glaucoma or optineurin-related An increased risk of glaucoma is diagnosed. The absence of degeneration in the optineurin gene indicates that the individual does not have glaucoma or degeneration associated with an increased risk of glaucoma, and thus this may cause optineurin-related glaucoma or optineurin. It is diagnosed that there is no associated increased risk of glaucoma.

  Allele-specific oligonucleotides are also used to detect the presence of denaturation in the optineurin gene by using dot blot hybridization of amplified oligonucleotides and allele-specific oligonucleotide (ASO) probes. (See, for example, Saiki, R. et al. (1986) Nature (London) 324: 163-166). An “allele-specific oligonucleotide” (also referred to herein as an “allele-specific oligonucleotide probe”) refers to an optineurin gene of about 10-50 base pairs, preferably about 15-30 base pairs. Oligonucleotides that specifically hybridize and contain modifications that are associated with glaucoma or an increased risk of glaucoma. Allele-specific oligonucleotide probes specific for a particular denaturation in the optineurin gene can be prepared using standard methods (see Current Protocols in Molecular Biology (supra)). Test samples of DNA are obtained from individuals to identify degeneration in genes that are associated with glaucoma or an increased risk of glaucoma. PCR can be used to amplify the entire or fragment of the optineurin gene and its flanking sequences. Using standard methods, DNA containing the amplified optineurin gene (or gene fragment) is dot blotted (see Current Protocols in Molecular Biology (supra)) and the blot is contacted with an oligonucleotide probe. The presence of specific hybridization of the probe to the amplified optineurin gene is then detected. Specific hybridization of an allele-specific oligonucleotide probe to DNA from an individual is an indicator of degeneration in the optineurin gene that is associated with glaucoma or an increased risk of glaucoma and is therefore associated with optineurin Is an indicator of increased risk of glaucoma or optineurin-related glaucoma. The absence of specific hybridization of an allele-specific oligonucleotide probe to DNA from an individual is an indication of the absence of degeneration in the optineurin gene associated with glaucoma or an increased risk of glaucoma Thus, it is an indication that there is no increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

  Other nucleic acid analysis methods can be used to detect denaturation in the optineurin gene. Representative methods include, for example, direct manual sequencing (Church and Gilbert, (1988), Proc. Natl. Acad. Sci. USA, 81: 1991-1995; Sanger, F. et al., (1977), Proc. Natl. Acad. Sci., 74: 5463-5467; Beavis et al., US Pat. No. 5,288,644); automated fluorescent sequencing; single-strand conformation denaturation assay (SSCP); fixed denaturing gel electrophoresis (CDGE) Denaturing gradient gel electrophoresis (DGGE) (Sheffield, VC et al. (19891), Proc. Natl. Acad. Sci. USA, 86: 232-236), mobility shift analysis (Orita, M. et al., (1989), Proc. Natl. Acad. Sci. USA, 86: 2766-2770), restriction enzyme analysis (Flavell et al., (1978), Cell, 15:25; Geever et al., (1981), Proc. Natl. Acad. Sci. USA, 78: 5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al., (1985), Proc. Natl. Acad. Sci. USA, 85: 4397-4401); RNase protection assay (Myers, RM et al. (1985), Science, 230: 1242); recognizes nucleotide mismatches such as E. coli mutS protein Use of the polypeptide; allele specific PCR is included.

Optineurin polypeptide-based methods In another embodiment of the invention, diagnosis of the presence or absence of an increased risk of optineurin-related glaucoma or optineurin-related glaucoma is that of an optineurin polypeptide. It can also be done by examining expression and / or composition. Test samples from individuals are evaluated for the presence or absence of altered expression and / or altered composition of the polypeptide encoded by the optineurin gene. The alteration in expression of the polypeptide encoded by the optineurin gene can be, for example, quantitative alteration of the polypeptide expression (ie, the amount of polypeptide produced), encoded by the optineurin gene. A modification of the composition of a polypeptide is a qualitative modification of polypeptide expression. Any such modification may be present. As used herein, “denaturation” of the expression or composition of a polypeptide refers to the alteration of the expression or composition of the test sample as compared to the expression or composition of the polypeptide by the optineurin gene in the control sample. A control sample is a sample from an individual that corresponds to the test sample (eg, from the same type of cells) and is not afflicted with glaucoma and has no increased risk of glaucoma. Altered expression or composition of the polypeptide in the test sample compared to the control sample is an indication of increased risk of optineurin-related or optineurin-related glaucoma. The absence of degeneration in the expression or composition of the polypeptide in the test sample compared to the control sample is an indication that there is no increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

  Expression of polypeptides encoded by the optineurin gene, including immunoassays (eg, David et al., US Pat. No. 4,376,110) such as spectroscopic, colorimetric, electrophoresis, isoelectric focusing, and immunoblotting. Alternatively, various means of examining the composition can be used (see also Current Protocols in Molecular Biology, especially Chapter 10). For example, polymorphism using Western blot analysis using an antibody that specifically binds to a polypeptide encoded by a mutated optineurin gene, or an antibody that specifically binds to a polypeptide encoded by a non-mutated gene. Alternatively, it can be identified that the polypeptide encoded by the mutant optineurin gene is present in the test sample, or that the polypeptide encoded by the non-polymorphic or non-mutated gene is not present in the test sample. The presence of a polypeptide encoded by a polymorphic or mutated gene, or the absence of a polypeptide encoded by a non-polymorphic or non-mutated gene is associated with an increased risk of glaucoma or glaucoma Therefore, this diagnoses an increased risk of optineurin-related glaucoma or optineurin-related glaucoma. The absence of a polypeptide encoded by a polymorphic or mutated gene or the presence of a polypeptide encoded by a non-polymorphic or non-mutated gene is associated with an increased risk of glaucoma or glaucoma Is therefore an indication that there is no optineurin-related glaucoma or an increased risk of optineurin-related glaucoma.

  In one embodiment of the method, the level or amount of polypeptide encoded by the optineurin gene in the test sample is compared to the level or amount of polypeptide encoded by the optineurin gene in the control sample. If the level or amount of polypeptide in the test sample is higher or lower than the level or amount of polypeptide in the control sample (though the difference is statistically significant), expression of the polypeptide encoded by the optineurin gene Is an indicator of degeneration in which it is diagnosed as an increased risk of optineurin-related glaucoma or optineurin-related glaucoma. If there is no statistical difference between the level or amount of polypeptide in the control sample and the level or amount of polypeptide in the test sample, there is no denaturation in the expression of the polypeptide encoded by the optineurin gene. It is diagnosed that there is no increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

  Alternatively, the composition of the polypeptide encoded by the optineurin gene in the test sample is compared to the composition of the polypeptide encoded by the optineurin gene in the control sample. A difference in the composition of the polypeptide in the test sample compared to the composition of the polypeptide in the control sample diagnoses an increased risk of optineurin-related glaucoma or optineurin-related glaucoma. There may be no increased risk of optineurin-related glaucoma or optineurin-related glaucoma due to the absence of a difference in the composition of the polypeptide in the test sample compared to the composition of the polypeptide in the control sample. Diagnosed.

  In another embodiment, both the level or amount of polypeptide and the composition of the test and control samples can be assessed. Differences in the amount or level of polypeptide in the test sample compared to the control sample; differences in composition of the test sample compared to the control sample; or both the difference in amount or level and the difference in composition may result in optineurin-related glaucoma or It is an indicator of increased risk of optineurin-related glaucoma. The absence of both quantity or level differences and compositional differences is an indication that there is no optineurin-related glaucoma or an increased risk of optineurin-related glaucoma.

Other Methods Based on Optineurin Polypeptides In another embodiment of the invention, a diagnosis of the presence or absence of an increased risk of optineurin-related glaucoma or optineurin-related glaucoma is optineurin poly It can also be performed by examining the activity of the peptide. Evaluation of the presence or absence of degeneration in the activity of the polypeptide encoded by the optineurin gene by comparing the test sample from the individual with the activity of the polypeptide encoded by the optineurin gene in the control sample To do. As described below and shown in FIG. 2, optineurin interacts with a variety of proteins including E3-14.7K, a component of the TNF-α pathway, and a component of the FAS ligand pathway. These proteins are referred to herein as “optineurin interacting polypeptides”.

  Altering the activity of a polypeptide encoded by an optineurin gene can be, for example, an increase or decrease in the interaction between an optineurin polypeptide and an optineurin interacting polypeptide. The level or amount of interaction of optineurin with an optineurin interacting polypeptide can be assessed in a test sample and a control sample (eg, a sample comprising a native optineurin polypeptide). The difference in the amount or level of interaction in the test sample compared to the control sample is an indicator of the presence of degeneration in optineurin and therefore the risk of optineurin-related or optineurin-related glaucoma It is an increase index. The absence of a difference in the amount or level of interaction is an indicator of the absence of such degeneration, and therefore there is no increased risk of optineurin-related or optineurin-related glaucoma. It is an indicator. For example, as described below, the C-terminal portion of optineurin interacts with huntingtin. Alteration in the amount or level of interaction between optineurin and huntingtin in the test sample compared to the amount or level of interaction between optineurin and huntingtin in the control sample It is an indicator of an increased risk of glaucoma or optineurin-related glaucoma.

  In another example, the amount or level of activity of an optineurin interacting polypeptide can be used as an indirect measure of the amount or level of optineurin activity. A difference in the amount or level of activity of the optineurin interacting polypeptide in the test sample compared to the control sample is an indicator of the presence of degeneration in the optineurin and therefore, optineurin-related glaucoma or optineurin It is an indicator of an increased risk of associated glaucoma. The absence of a difference in the amount or level of activity of the optineurin-interacting polypeptide is an indicator of the absence of such degeneration, and therefore, the risk of optineurin-related glaucoma or optineurin-related glaucoma. It is an indicator that there is no increase. For example, as described below, optineurin can inhibit the production of TNF-α (either directly or through interaction with other proteins). Thus, the amount of TNF-α production can be assessed and can be used as a surrogate for the amount of optineurin activity. Alteration of the amount or level of TNF-α in the test sample compared to the amount or level of TNF-α in the control sample (for example, an increase in the amount of TNF-α) is an indication of the presence of an optineurin mutation. Thus, it is an indication that there is an increased risk of optineurin-related glaucoma or optineurin-related glaucoma.

Kits useful for diagnostic methods include, for example, hybridization probes, restriction enzymes (e.g. for RFLP analysis), allele-specific oligonucleotides, antibodies that bind to mutated or non-mutated (native) optineurin polypeptides, optineurin genes Components useful in any of the methods described herein, including means for amplifying the nucleic acid comprising, or means for analyzing the nucleic acid sequence of an optineurin gene or analyzing the amino acid sequence of an optineurin polypeptide, etc. Including.

Therapeutic methodsThe present invention also includes methods of treating an increased risk of glaucoma or glaucoma (prophylactic and / or therapeutic methods), and treatment of an increased risk of glaucoma or glaucoma using an optineurin therapeutic. It also relates to the use of an optineurin therapeutic agent for the manufacture of a pharmaceutical product. This method can be used not only in individuals who have been diagnosed or suspected of having an increased risk of optineurin-related glaucoma or optineurin-related glaucoma but is not related to optineurin It can also be used in individuals who have been diagnosed or suspected of having an increased risk of glaucoma or glaucoma. This is because this method can be equally useful in such individuals by modifying the course of glaucoma. As described below and shown in FIG. 2, optineurin interacts with a variety of proteins including E3-14.7K, a component of the TNF-α pathway, and a component of the FAS ligand pathway. These proteins, called “optineurin-interacting polypeptides”, are suitable targets for optineurin therapeutics to modify glaucoma by altering the activity and interaction between them and optineurin.

  An “optineurin therapeutic” is an agent used to treat glaucoma (eg, an optineurin agonist) that denatures (eg, enhances or inhibits) optineurin polypeptide activity and / or optineurin gene expression. Or antagonist). This therapy is designed to inhibit, denature, replace or supplement the activity of an optineurin polypeptide in an individual, or to inhibit, denature, replace or supplement the activity of an optineurin interacting polypeptide in an individual.

  Optineurin therapeutics, for example, provide additional proteins or upregulate transcription or translation of optineurin; modify post-translational processing of optineurin polypeptides; transcription of splicing variants of optineurin Various; such as modifying the activity of an optineurin polypeptide (e.g. by binding to optineurin) or altering the transcription or translation of optineurin (up-regulating or down-regulating) By such means, the activity of optineurin or gene expression can be altered. Other optineurin therapeutics use optineurin-interacting polypeptides to alter the activity or expression of genes encoding optineurin-interacting polypeptides or other genes in pathways involving optineurin. Can be targeted.

  Exemplary optineurin therapeutics include several different drug classes.

In one embodiment, an optineurin therapeutic encodes a gene, cDNA, mRNA, a nucleic acid encoding an optineurin polypeptide (e.g., SEQ ID NO: 1, 3, or 5), or a variant of optineurin. A nucleic acid, such as a nucleic acid encoding a variant (mutant nucleic acid molecule does not necessarily have to exist in nature, but encodes a polypeptide having the amino acid sequence of optineurin). Can be. Thus, for example, a DNA molecule comprising a sequence that differs from a naturally occurring nucleotide sequence but encodes optineurin due to the degeneracy of the genetic code is contemplated and encodes a portion (fragment) or optineurin Nucleotide sequences that encode variant polypeptides such as analogs or derivatives of are also contemplated. Such variants are naturally occurring, as is the case with allelic variations or single nucleotide polymorphisms, or naturally occurring, such as those induced by various mutagens or mutagenic processes. Can not be. Contemplated mutations include, but are not limited to, one or more nucleotide additions, deletions and substitutions that can result in conservative or non-conservative amino acid changes, including additions and deletions. Preferably, nucleotide (and / or resulting amino acid) changes are silent or conserved, i.e., this allows optineurin characteristics or activity (e.g., interacts with other specific proteins as detailed below). Does not change. Other modifications of nucleic acid molecules include, for example, labeling, methylation, and uncharged bonds (eg, methylphosphonates, phosphotriesters, phosphoamidates, carbamates), charged bonds (eg, phosphorothioates, phosphorodithioates), pendant moieties Intranucleotide modifications such as (eg, polypeptides), intercalating agents (eg, acridine, psoralen), chelating agents, alkylating agents, and modified linkages (eg, alpha anomeric nucleic acids) can be included. Also included are synthetic molecules that mimic nucleic acid molecules with respect to their ability to bind to sequences designated by hydrogen bonding and other chemical interactions. Such molecules include, for example, molecules in which phosphate bonds are replaced with peptide bonds in the main chain of the molecule.

  Other optineurin therapeutics include aptamers, which are DNA or RNA molecules selected based on their ability to bind to other molecules (see, for example, the aptamer database at aptamer.icmb.utexas.edu/; Gening See also, LV et al., Biotechniques, 31 (4): 828, 830, 832, 834, (2001).

Proteins and polypeptidesIn another embodiment, the optineurin therapeutic is encoded by an optineurin polypeptide (e.g., SEQ ID NOs: 2, 4, and 6), a peptidomimetic, or a derivative of an optineurin polypeptide, or an optineurin gene. It can be another splicing variant or a fragment or derivative thereof. Fusion proteins or other polypeptides that contain fragments (particularly those that retain the activity of optineurin) can be used, including optineurin polypeptides that include sequencing variants Can do.

  An active fragment performs one or more of the same functions as an all optineurin polypeptide (the ability to interact with other specific proteins, as described below). For example, an active fragment is a domain, segment, or motif that has been identified by analysis of protein sequences using well-known methods, eg, signal peptides, extracellular domains, one or more transmembrane segments or loops, ligand binding A region, Zn finger domain, DNA binding domain, acylation site, glycosylation site, or phosphorylation site can be included. Active fragments can be individual (not fused to other amino acids or polypeptides) or within a larger polypeptide. In addition, several fragments can be contained within a single larger polypeptide.

  Variants include substantially homologous polypeptides encoded at the same locus in an organism, ie allelic variants, as well as other splicing variants. Variants also include polypeptides that are derived from other genetic loci in the organism but have significant homology to the above-described polypeptides encoded by the optineurin gene or nucleic acid. Variants also include proteins that are substantially homologous or identical to these proteins, but from other organisms, ie, homologous molecular species. Variants also include proteins that are substantially homologous or identical to these proteins but that are produced by chemical synthesis. Variants also include proteins that are substantially homologous or identical to these proteins and that are produced by recombinant methods. Similarity is determined by conservative amino acid substitutions. Such a substitution is a substitution in which a predetermined amino acid in a polypeptide is substituted with another amino acid having similar characteristics. Conservative substitutions are likely to be phenotypically silent. Typically seen as conservative substitutions, substitution of one of the aliphatic amino acids Ala, Val, Leu and Ile with another; replacement of hydroxyl residues Ser and Thr, exchange of acidic residues Asp and Glu Substitution between the amide residues Asn and Gln, exchange of the basic residues Lys and Arg, and substitution between the aromatic residues Phe and Tyr. Guidance on which amino acid changes are phenotypically silent can be found in Bowie et al., Science, 247: 1306-1310, (1990). A variant polypeptide can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or any combination thereof. Furthermore, variant polypeptides can be fully functional or lack function with one or more activities. Fully functional variants typically include only conservative mutations or mutations in non-lethal residues or non-lethal regions. Functional variants can also include substitutions of similar amino acids that result in no change or insignificant change in function. Alternatively, such substitutions may have some positive or negative impact on function. Non-functional variants are typically one or more non-conservative amino acid substitutions, deletions, insertions, inversions, or truncations, or substitutions, insertions, inversions of critical residues or critical regions Or a deletion. Amino acids important for function can be identified by methods well known in the art such as site-directed mutagenesis and alanine scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085, (1989)). Sites important for polypeptide activity can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol., 224: 899-904, (1992 de Vos et al., Science 255: 306-312 (1992)).

The optineurin polypeptides and other polypeptides described herein can be isolated from naturally occurring sources, chemically synthesized, or recombinantly produced. For example, the nucleic acid molecules described herein can be used to generate recombinant forms of proteins encoded by microbial or eukaryotic cell processes. Ligating a polynucleotide sequence into a gene construct such as an expression vector, transforming or transfecting into either a eukaryotic (yeast, bird, insect, plant, mammal) or prokaryotic (bacterial cell) host Are standard procedures used to produce other well-known proteins. Recombinant proteins can be prepared by microbial means or tissue culture techniques using similar or modified procedures. Protein ions can be isolated or purified from cell culture (eg, until homogeneous) by various methods. These include, but are not limited to, anion or cation exchange chromatography, ethanol precipitation, affinity chromatography, and high performance liquid chromatography (HPLC). The specific method used will depend on the properties of the protein, and appropriate methods will be readily understood by those skilled in the art. For example, with respect to protein or protein identification, bands identified by gel analysis can be purified by HPLC, and the resulting purified protein can be sequenced. Alternatively, purified proteins can be purified enzymatically by methods known in the art to purify protein fragments and sequenced. Sequencing can be performed, for example, by the method of Wilm et al. (Nature, 379 (6564): 466-469, (1996)). Protein, Jacoby, Methods in Enzymology, 104, Academic Press, New York, (1984); Scopes, Protein Purification, Principles and Practice, 2nd edition, Springer-Verlag, New York, (1987); and Deutscher , Guide to Protein Purification, Methods in Enzymol
isolated by conventional means of protein biochemistry and purification, as described in ogy, Vol. 182, (1990) and is substantially pure, ie, contains only 20, 5 or 1% of cellular component impurities A product can be obtained.

Antibodies and other small moleculesIn another embodiment, the optineurin therapeutic is an antibody (e.g., an antibody to a mutant optineurin polypeptide, an antibody to a non-mutant optineurin polypeptide, or a specific splicing variant of an optineurin polypeptide). Antibodies); ribozymes; peptidomimetics; small molecules or other agents that modify optineurin polypeptide activity and / or gene expression (e.g., those that upregulate or downregulate optineurin gene expression); or Alter (eg, enhance or inhibit) optineurin gene expression or optineurin polypeptide activity, alter post-translational processing of optineurin polypeptide, or optineurin splicing Another agent which modulates transcription of variants can be (e.g., which drugs splice variant affects either expressed or agents that affect the amount of each splice variant is expressed,).

For example, an antibody against a mutant optineurin polypeptide can be used to inhibit the activity of the mutant protein. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, ie, molecules that contain an antigen binding site that specifically binds an antigen. A molecule that specifically binds to a polypeptide is a molecule that binds to a polypeptide or fragment thereof but does not substantially bind to other molecules in a sample, eg, a biological sample that naturally contains the polypeptide. Examples of immunologically active portions of immunoglobulin molecules include F (ab) and F (ab ′) ₂ fragments that can be generated by treating an antibody with an enzyme such as pepsin. Either polyclonal or monoclonal antibodies can be used. As used herein, the term “monoclonal antibody” or “monoclonal antibody composition” refers to an antigen binding site that has the ability to immunoreact with a particular epitope of one protein (eg, mutant optineurin). It refers to a group of antibody molecules containing only species. Accordingly, a monoclonal antibody composition typically displays a single binding affinity for a particular polypeptide with which it undergoes an immune response.

  Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a desired immunogen, such as an optineurin polypeptide or a fragment thereof. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as an enzyme linked immunosorbent assay (ELISA) using immobilized polypeptide. If desired, antibody molecules against the polypeptide can be isolated from a mammal (eg, from blood) and further purified by well-known techniques such as protein A chromatography to obtain an IgG fraction. After an appropriate time after immunization, for example when the antibody titer is highest, antibody-producing cells are obtained from the subject and first described in Kohler and Milstein, (1975) Nature, 256: 495-497. Hybridoma technology, human B-cell hybridoma technology (Kozbor et al. (1983), Immunol. Today, 4:72), EBV hybridoma technology (Cole et al. (1985), Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. ., Pp. 77-96) or can be used to prepare monoclonal antibodies by standard techniques such as trioma technology. Techniques for generating hybridomas are well known (see generally, Current Protocols in Immunology, (1994), Coligan et al., John Wiley & Sons, Inc., New York, NY). Briefly, an immortalized cell line (typically myeloma) is fused with lymphocytes (typically splenocytes) from a mammal immunized with an immunogen as described above and the resulting hybridoma cells The culture supernatant is screened to identify hybridomas producing monoclonal antibodies that bind to the protein of interest.

  Any of a number of well-known protocols used for fusion of lymphocytes with immortalized cell lines can be applied to produce monoclonal antibodies to optineurin polypeptides (e.g., Current Protocols in Immunology ( Galfre et al., (1977) Nature, 266: 55052; RH Kenneth, Monoclonal Antibodies: A New Dimension In Biological Analyses, Plenum Publishing Corp., New York, New York, (1980); and Lerner, (1981). Yale J. Biol. Med., 54: 387-402 Further, those skilled in the art will appreciate that there are many variations of such methods that are also useful.

  Instead of preparing a hybridoma that secretes a monoclonal antibody, a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) is screened with the polypeptide, thereby isolating immunoglobulin library members that bind to the polypeptide. Can identify and isolate monoclonal antibodies. Kits for generating and screening phage display libraries are commercially available (eg, Pharmacia's Recombinant Phage Antibody System, catalog number 27-9400-01; and Stratagene's SurfZAP ™ phage display kit, catalog number 240612). In addition, examples of methods and reagents that are particularly accepted for use in generating and screening antibody display libraries include, for example, U.S. Patent No. 5,223,409; International Publication No. WO 92/18619; International Publication No. WO 91/17271; International Publication WO 92/20791; International Publication WO 92/15679; International Publication WO 93/01288; International Publication WO 92/01047; International Publication WO 92/09690; International Publication WO 90 / 02809; Fuchs et al. (1991) Bio / Technology 9: 1370-1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3: 81-85; Huse et al. (1989) Science 246 : 1275-1281; found in Griffiths et al. (1993), EMBO J., 12: 725-734.

  In addition, recombinant antibodies can be used, such as chimeric and humanized monoclonal antibodies that contain both human and non-human portions that can be made using standard recombinant DNA techniques. Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art.

In a fourth embodiment, an optineurin therapeutic agent is a polypeptide that interacts with optineurin ("optineurin interacting polypeptide"); encodes such a polypeptide that interacts with optineurin. An agent that modifies the expression or activity of the optineurin interacting polypeptide; and / or an agent that modifies the interaction between the optineurin and the optineurin interacting polypeptide. For example, as detailed below and shown in FIG. 2, optineurin is a protein associated with the FAS ligand pathway and a protein associated with the TNF-α pathway (eg, huntingtin, caspase 9), as well as RAB-8, TFIIIA , And interact with proteins related to E3-14.7K. Optineurin further interacts with cytoplasmic phospholipase and cytochrome P450. Furthermore, optineurin is believed to act with TNF-α through a feedback mechanism, thereby playing a neuroprotective role in optic neuropathy. Thus, the expression of any one of these polypeptides that interacts with optineurin is denatured (increased or decreased), thereby altering the amount of optineurin activity, which in turn results in the neuroprotective action of optineurin. Can be used to augment roles. For example, agents that regulate TNF-α production or TNF- in terms of the feedback mechanisms of optineurin and TNF-α, and in terms of the increase in TNF-α found in patients with glaucoma Agents that reduce the amount of α will function in a manner similar to optineurin, ie, reduce TNF-α and act as neuroprotection against the effects of TNF-α. Thus, in certain embodiments, the agent is an agent that modifies the expression of TNF-α. The agent that modifies the expression or activity of an optineurin interacting polypeptide can be, for example, any type of agent described herein (eg, a nucleic acid, polypeptide or protein, antibody, etc.).

  If desired, multiple optineurin therapeutics can be used simultaneously. Optineurin therapeutics can be used in therapeutically effective amounts (i.e., to improve symptoms associated with the disease, to prevent or delay the onset of the disease (e.g., particularly in individuals with an increased risk of glaucoma), and / or Or in an amount sufficient to treat the disease, such as by reducing the severity or frequency of the disease symptoms. The therapeutically effective amount for treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be used to help identify optimal dosage ranges. The exact dosage used in the formulation will also depend on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  As used herein, the term “treatment” refers not only to ameliorating symptoms associated with a disease, but also to preventing or delaying the onset of the disease, as well as reducing the severity or frequency of the symptoms of the disease. Thus, as used herein, “treatment of glaucoma” refers not only to treatment after the appearance of glaucoma symptoms (therapeutic treatment), but also to prophylactic treatment (before the appearance of symptoms). Treatment may be particularly useful in individuals who have been identified with an increased risk of glaucoma because they can delay the onset of the disease or completely prevent the symptoms of the disease. Thus, treatment is not limited to individuals suffering from glaucoma but also to individuals at risk of developing glaucoma (e.g., individuals with degeneration in the optineurin gene that are associated with an increased risk of glaucoma). It can also be used for individuals with increased risk.

  In one embodiment of the invention, the nucleic acid is used to treat glaucoma. The nucleic acids described above can be used alone or in a pharmaceutical composition as described above. For example, a cDNA encoding an optineurin gene or an optineurin polypeptide alone or in a vector so that the cell produces a native optineurin polypeptide (in vitro or in vitro). Can be introduced. In another example, a gene encoding an optineurin-interacting polypeptide or a cDNA encoding an optineurin-interacting polypeptide, alone or in a vector, allows the cell to contain a native optineurin-interacting polypeptide. It can be introduced into cells to produce peptides (in vitro or in vivo). If necessary, cells transformed with a gene or cDNA or a vector containing the gene or cDNA can be introduced (or reintroduced) into an individual suffering from a disease. Accordingly, cells that are inherently lacking native expression and activity of the polypeptide, or cells that have mutated expression and activity, can be engineered to produce the desired polypeptide (e.g., optineurin polypeptide, or e.g., optineurin). Active fragments of polypeptides) can be expressed. In a preferred embodiment, a nucleic acid encoding an optineurin polypeptide or an active fragment or derivative thereof is introduced into an expression vector, such as a viral vector, which is suitable for lacking expression of the animal's native optineurin. It can be introduced into cells. For example, for the treatment of glaucoma, a vector containing the nucleic acid can be introduced into the eye. In such methods, the population of cells can be genetically engineered such that the active optineurin polypeptide is expressed by induction or constitutively. Other genes including viral and non-viral transfer systems can be used. Alternatively, non-viral gene transfer methods such as calcium phosphate coprecipitation, mechanical techniques (eg microinjection); membrane fusion mediated transfer by liposomes; or direct DNA uptake can also be used.

  Alternatively, in another embodiment of the present invention, the nucleic acid described above or a nucleic acid complementary to such a nucleic acid is replaced with mRNA and / or genomic DNA of an optineurin gene (or a gene encoding an optineurin interacting polypeptide). (Ie, mRNA and / or genomic DNA) can be used in “antisense” therapy where nucleic acids (eg, oligonucleotides) that specifically hybridize are administered or generated in situ. Antisense nucleic acids that specifically hybridize to mRNA and / or DNA inhibit expression of an optineurin polypeptide or an optineurin interacting polypeptide, for example, by inhibiting translation and / or transcription. Antisense nucleic acid binding can be by conventional base-pair complementarity, or by specific interaction of the main groove of the double helix, for example, in the case of binding to a DNA duplex.

  The antisense construct can be delivered as an expression plasmid, eg, as described above. When the plasmid is transcribed in the cell, mRNA that encodes the optineurin polypeptide (or optineurin interacting polypeptide) and / or RNA complementary to a portion of the DNA is produced. Alternatively, the antisense construct can be an oligonucleotide probe that is generated ex vivo and introduced into the cell. The antisense construct then inhibits expression by hybridizing with mRNA and / or genomic DNA. In one embodiment, the oligonucleotide probes are modified oligonucleotides that are resistant to endogenous nucleases, such as exonucleases and / or endonucleases, and thus are stable in vivo. Examples of nucleic acid molecules used as antisense oligonucleotides are phosphoramidate, phosphothioate, and methylphosphonate analogs of DNA (see US Pat. Nos. 5,176,996; 5,264,564; and 5,256,775). In addition, common approaches to construct oligomers useful for antisense therapy include, for example, Van der Krol et al. ((1988), Biotechniques, 6: 958-976); and Stein et al. ((1988), Cancer Res, 48: 2659-2668). For antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, eg, the region between -10 and +10 of the optineurin gene sequence, are preferred.

  To perform antisense therapy, an oligonucleotide (mRNA, cDNA or DNA) complementary to the mRNA encoding optineurin (or optineurin interacting polypeptide) is designed. Antisense oligonucleotides bind to mRNA transcripts and prevent translation. Absolute complementarity is preferred but not essential. As used herein, a sequence that is “complementary” to a portion of RNA refers to that the sequence has sufficient complementarity to hybridize with RNA to form a stable duplex. In the case of double-stranded antisense nucleic acids, one strand of the double-stranded DNA can be tested in this way, or triplex formation can be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid as described above. In general, the longer a nucleic acid that hybridizes, the more it can contain a base mismatch with RNA, but still form a stable duplex (or possibly triplex). One skilled in the art can ascertain the degree of mismatch allowed by using standard procedures.

  The nucleic acid molecule used to form a triple helix to inhibit transcription is preferably single stranded and consists of deoxyribonucleotides. The base composition of such oligonucleotides facilitates the formation of triple helices by Hogsteen's base-pairing law, which generally requires the presence of a rather large stretch of either purines or pyrimidines in one strand of the duplex. Should do. The nucleotide sequence may be based on pyrimidines, resulting in TAT and CGC triplets across the three related strands of the resulting triple helix. A pyrimidine-rich molecule provides base complementarity to one purine-rich region of a double-stranded chain in a direction parallel to the chain. In addition, purine-rich nucleic acid molecules, such as nucleic acid molecules containing stretches of G residues, may be selected. Such molecules form a triple helix with a heavy chain in a GC pair-rich DNA, with the majority of purine residues located on one strand of the target duplex, spanning three strands of the triplex. Bring CGC triplets. A potential sequence that can be targeted for triple helix formation is first base paired with one strand of the duplex and then with the other strand, resulting in two rather large stretches of either purines or pyrimidines. Increased by creating a “switchback” nucleic acid molecule in which 5′-3 ′ and 3′-5 ′ are synthesized in an alternating fashion so that the need to be present on one strand of the heavy chain is eliminated can do.

  In a preferred embodiment, an oligonucleotide complementary to the 5 ′ end of the message, eg, from the 5 ′ untranslated sequence to where it contains the AUG start codon, is used to inhibit translation. However, it has recently been shown that sequences complementary to the 3 'untranslated sequence of mRNA are also effective in inhibiting translation of mRNA (Wagner, R., (1994), Nature, 372: 333). Thus, oligonucleotides complementary to either the 5 'or 3' untranslated noncoding region of the optineurin gene (or the gene encoding the optineurin interacting polypeptide) also inhibit translation of endogenous mRNA. It can be used in an antisense approach. An oligonucleotide complementary to the 5 ′ untranslated region of the mRNA can include a sequence complementary to the AUG start codon. Antisense oligonucleotides complementary to the mRNA coding region can also be used in accordance with the present invention. Antisense nucleotides complementary to the can region sequence can be used, but those complementary to the transcribed untranslated region can also be used. Regardless of whether it is designed to hybridize to the 5 'region, 3' region, or coding region of an optineurin mRNA, the antisense nucleic acid is preferably at least 6 nucleotides in length, more preferably in length. Oligonucleotides ranging from 6 to about 50 nucleotides. In certain preferred embodiments, the oligonucleotide is at least 10 nucleotides, at least 18 nucleotides, at least 24 nucleotides, or at least 50 nucleotides.

  If desired, in vitro studies can be performed to quantify the ability of antisense oligonucleotides to inhibit gene expression. Such studies utilize controls that distinguish between gene inhibition by oligonucleotide antisense and non-specific biological effects. In these studies, the level of target RNA or protein can be compared to the level of internal control RNA or protein. In a preferred embodiment, the control oligonucleotide is approximately the same length as the test oligonucleotide, and the nucleotide sequence of the oligonucleotide differs from the antisense sequence sufficient to prevent specific hybridization to the target sequence.

  Oligonucleotides used in antisense therapy can be single-stranded or double-stranded, DNA, RNA, chimeric mixtures or derivatives thereof or modifications thereof. Oligonucleotides can be modified in the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization, and the like. Oligonucleotides can be other appending groups such as peptides (eg to target host cells in vivo) or cell membranes (eg Letsinger et al. (1989), Proc. Natl. Acad. Sci. USA, 86: 6553). -6556; Lemaitre et al., (1987), Proc. Natl. Acad Sci. USA, 84: 648-652; see WO 88/09810) or the blood brain barrier (eg, WO 89/10134). Agents that facilitate transport through, or cleavage agents that act upon hybridization (see, for example, Krol et al. (1988), BioTechniques, 6: 958-976) or intercalating agents (see, for example, Zon, (1988) ), Phare. Res., 5: 539-549). To this end, the oligonucleotide can be conjugated to another molecule, such as a peptide, a cross-linking agent that operates by hybridization, a transport agent, a cleavage agent that operates by hybridization, and the like.

  Antisense oligonucleotides include, but are not limited to, 5-fluorouracil, 5-uracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxymethyl) uracil, 5 -Carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcueosin (queosine), inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2 -Thiouracil, β-D-mannosylcueosin, 5'-methoxycarboxymethylura , 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, cueosin, 2-thiocytosine, 5-methyl-2- Thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3- (3-amino-3 At least one (or more) modified base moiety selected from the group comprising -N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine can be included. Antisense oligonucleotides can also include at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose. In another embodiment, the antisense oligonucleotide consists of phosphorothioate, phosphorodithioate, phosphoramidothioate, phosphoramidate, phosphordiamidate, methylphosphonate, alkylphosphotriester, and formacetal or analog thereof At least one modified phosphate backbone selected from the group. In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. α-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNAs, in which the strands are parallel to each other as opposed to normal β units (Gautier et al. (1987) Nucl. Acids Res ., 15: 6625-6641). Oligonucleotides can be 2′-O-methyl ribonucleotides (Inoue et al. (1987), Nucl. Acids Res., 15: 6131-6148), or chimeric RNA-DNA analogs (Inoue et al. (1987), FEBS Lett ., 215: 327-330).

  Oligonucleotides can be synthesized by standard methods described herein that are well known in the art (eg, those commercially available from automated DNA synthesizers (such as those commercially available from Biosearch, Applied Biosystems, etc.). Phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. ((1988) Nucl. Acids Res. (Sarin et al., (1988), Proc. Natl. Acad. Sci. USA., 85: 7448-7451).

  Antisense molecules are delivered in vivo into cells that express optineurin (or an optineurin interacting polypeptide). Several methods can be used to deliver antisense DNA or RNA to cells. For example, antisense molecules can be injected directly into a tissue site, or modified antisense molecules designed to target the desired cells (eg, peptides or antibodies that specifically bind to the receptor or target cell surface An antisense molecule linked to the antibody expressed above can be administered systemically. Alternatively, a preferred embodiment utilizes a recombinant DNA construct that places the antisense oligonucleotide under the control of a strong promoter (eg, polIII or polII). Using such a construct to transfect target cells into a patient, a sufficient amount of single-stranded RNA that forms complementary base pairs with the endogenous transcript is transcribed, thus translating mRNA. Is disturbed. For example, a vector can be introduced in vivo such that it is taken up into a cell and directs transcription of an antisense RNA. Such a vector may remain episomal or be integrated into the chromosome so long as it can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA techniques described above that are standard in the art. For example, a plasmid, cosmid, YAC or viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site (eg, intraocular tissue). Alternatively, viral vectors that selectively infect the desired tissue can be used, in which case administration may be by another route (eg, systemic administration).

  Ribozyme molecules designed to catalytically cleave optineurin mRNA transcripts are also used to prevent translation of optineurin mRNA and expression of optineurin polypeptides, particularly for example, to prevent translation of mutant optineurin polypeptides (See, eg, International Publication No. WO 90/11364, and Sarver et al. (1990) Science 247: 1222-1225). Alternatively, they can be designed to catalytically cleave mRNA transcripts of genes encoding optineurin interacting polypeptides. Ribozymes are enzymatic RNA molecules that have the ability to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of ribozyme molecules to complementary target RNA, followed by cleavage within the nucleotide strand. The composition of the ribozyme molecule must include one or more sequences complementary to the target gene mNRA and must include a catalytic sequence responsible for mRNA cleavage. See US Pat. No. 5,093,246 for this sequence. Specific ribozyme cleavage sites within any potential RNA target are first identified by examining the molecule of interest for ribozyme cleavage sites including the following sequences: GUA, GUU and GUC. After identification, a short RNA sequence of about 15 to 20 ribonucleotides corresponding to the region of the target gene that contains the cleavage site, for expected structural features such as secondary structure that renders the oligonucleotide sequence inappropriate Can be evaluated. The suitability of a candidate sequence can also be assessed by testing its accessibility in hybridization with a complementary oligonucleotide using a ribonuclease protection assay. A ribozyme that cleaves mRNA at the site-specific recognition sequence can be used to destroy optineurin mRNA. In another embodiment, a hammerhead ribozyme is used. The hammerhead ribozyme cleaves mRNA at a position indicated by a flanking region that forms a complementary base pair with a target mRNA having a two-base sequence, 5′-UG-3 ′. The construction and production of hammerhead ribozymes is more fully described in Haseloff and Gerlach ((1988) Nature, 334: 585-591). Ribozymes must be genetically engineered so that the cleavage recognition site is located near the 5 'end of the optineurin mRNA to increase efficiency and minimize intracellular accumulation of nonfunctional mRNA transcripts. preferable.

  Ribozymes used in the present invention have been extensively described by Thomas Cech and co-workers (Zaug et al. (1984) Science 224: 574-578; Zaug and Cech (1986) Science 231 : 470-475; Zaug et al. (1986) Nature 324: 429-433; International Publication WO 88/04300; Been and Cech, (1986) Cell 47: 207-216), Tetrahymena tlierrnophila. RNA endoribonucleases (hereinafter “Cech-type ribozymes”) such as those naturally occurring (known as IVS or L-19 IVS RNA) can also be included. The Cech ribozyme has an active site of 8 base pairs that hybridizes with the target RNA sequence, and the target RNA is cleaved after hybridization. The invention further encompasses Cech-type ribozymes that target an active site of 8 base pairs present in optineurin.

  Similar to antisense approaches, ribozymes can be composed of modified oligonucleotides (e.g., improved stability, targeting, etc.) and delivered to cells expressing optineurin in vivo (e.g., ocular cells). . A preferred method of delivery is a ribozyme that is under the control of a strong constitutive promoter such that a sufficient amount of ribozyme is produced by the transfected cells to destroy endogenous messages and inhibit translation. Including the use of "coding" DNA constructs. Ribozymes are catalytic, unlike antisense molecules, so only low intracellular concentrations are required for good efficiency.

  Endogenous optineurin gene expression, particularly mutant optineurin gene expression, inactivates the optineurin gene or its promoter, or the optineurin-interacting polypeptide gene or promoter using targeted homologous recombination That is, it can also be reduced by `` knocking out '' (e.g. Smithies et al., (1985) Nature, 317: 230-234; Thomas and Capecchi, (1987) Cell, 51: 503-512; Thompson et al., (1989), Cell, 5: 313-321). For example, to transfect cells expressing optineurin in vivo, DNA homologous to the endogenous optineurin gene (optineurin) with or without a selectable marker and / or a negative selectable marker. Non-functional optineurin genes (or DNA sequences that are not related at all) can be used, which are flanked by either the coding region of the gene or regulatory sequences. Insertion of the DNA construct by homologous recombination results in inactivation of the optineurin gene. Similar methods can be used for genes encoding optineurin interacting polypeptides. The recombinant DNA construct can be administered or targeted directly to the required site in vivo using an appropriate vector, as described above. Alternatively, the expression of a non-mutated optineurin or optineurin interacting polypeptide can be increased using similar methods. That is, as described above, targeted homologous recombination can be used to insert a DNA construct containing a non-mutated functional gene into a cell instead of a mutated gene.

  Alternatively, the expression of an endogenous optineurin gene, or the expression of a gene encoding an optineurin interacting polypeptide targets a deoxyribonucleotide sequence complementary to the gene's regulatory sequence (i.e., promoter and / or enhancer). Can be reduced in the body by forming a triple helix structure that prevents transcription of the optineurin gene in the target cell (generally Helene, C., (1991), Anticancer Drug Des., 6 (6): 569-84; Helene, C. et al. (1992), Ann. NY Acad. Sci., 660: 27-36; and Maher, LJ, (1992), Bioassays, 14 (12): 807-15). Similarly, antisense constructs are used in tissue manipulation, such as tissue differentiation of both in vivo and ex vivo tissue cultures, by antagonizing one normal biological activity of an optineurin polypeptide. be able to. In addition, antisense techniques (e.g., microinjection of antisense molecules, or transfection with plasmids whose transcripts are antisense with respect to mRNA or gene sequence of optineurin) can be used to optimize optineurin in developmental events. As well as the normal cellular function of optineurin in adult tissues. Such techniques can be used in cell culture, but can also be used in the production of transgenic animals.

  In yet another embodiment of the invention, a polypeptide and / or agent that denatures (eg, enhances or inhibits) the activity of an optineurin polypeptide, as described herein, is used to treat or prevent glaucoma. Can be used for As described herein, polypeptides and / or agents that denature (eg, enhance or inhibit) the activity of an optineurin-interacting polypeptide can also be used to treat or prevent glaucoma. The polypeptide or agent can be delivered as a composition as described above or can be delivered alone. They can be administered systemically or can be targeted to specific tissues (eg, ocular tissues). The protein and / or agent may be produced by a variety of means including, for example, chemical synthesis; recombinant production; in vivo production (e.g., transgenic animals such as Meade et al., U.S. Pat.No. 4,873,316). And can be isolated using standard means as described herein.

  Any combination of the above treatment methods (eg, administration of a non-mutated optineurin polypeptide in combination with antisense therapy targeting mutant optineurin mRNA) can also be used.

Compositions for Treatment Methods The treatment methods described above utilize a drug that can be incorporated into a pharmaceutical composition if desired. For example, a physiologically acceptable nucleotide or nucleic acid construct (vector) containing a protein or protein, fragment, fusion protein or prodrug thereof, or a nucleic acid encoding optineurin or an agent that modifies the activity of optineurin. A pharmaceutical composition can be prepared by blending with a carrier or excipient. The carrier and composition can be sterile. The formulation should suit the mode of administration.

  Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions (eg, NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, benzyl alcohol , Polyethylene glycol, gelatin, lactose, carbohydrates such as amylose and starch, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid ester, hydroxymethylcellulose, polyvinylpyrrolidone and the like, and combinations thereof. If desired, the drug does not adversely react with the active compound, for example, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, coloring It can be mixed with flavoring agents, flavoring agents and / or aromatic substances.

  If desired, the composition can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can be a solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. This composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, polyvinyl pyrrolidone, sodium saccharinate, cellulose, magnesium carbonate.

  Methods for introducing these compositions include, but are not limited to, intradermal, intramuscular, intraperitoneal, intraocular, intravenous, subcutaneous, topical, oral and intranasal. In preferred embodiments, the composition is introduced into the eye (eg, eye drops). Other suitable methods of introduction may also include gene therapy (described below), rechargeable or biodegradable devices, particle accelerators (“gene guns”) and sustained release polymer devices. The pharmaceutical composition can also be administered as part of a combinatorial therapy with other drugs.

  The composition can be formulated according to conventional procedures as a pharmaceutical composition adapted for human administration. For example, a composition for intravenous administration is usually a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic to ease pain at the site of the injection. In general, the ingredients are individually formulated as a unit dosage form, eg, a dry lyophilized powder or a water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. Or mix and supply. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. Where the composition is administered by injection, an ampoule of sterile water or saline can be provided so that the ingredients may be mixed prior to administration.

  For topical application, a non-sprayed form from viscous to semi-solid or solid can be used, including a carrier that is compatible with local application and preferably has a dynamic viscosity greater than water. . Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, enemas, lotions, sols, liniments, salves, aerosols, etc. If desired, include those that are sterilized or mixed with adjuvants such as preservatives, stabilizers, wetting agents, buffers or salts to affect osmotic pressure, for example. Drugs may be incorporated into cosmetic formulations. For topical application, the active ingredient is packaged in a squeeze bottle, preferably with a solid or liquid inert carrier material, or a pressurized volatile ingredient, usually a gaseous propellant such as an additive. Also suitable are nebulizable aerosol preparations mixed with pressurized air.

  The agents described herein can be formulated as neutral or salt forms. Pharmaceutically acceptable salts include salts formed with free amino groups, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, and the like, sodium, potassium, ammonium, calcium, ferric hydroxide, isopropyl Included are salts formed with free carboxyl groups such as those derived from amines, triethylamine, 2-ethylaminoethanol, histidine, procaine and the like.

  The drug is administered in a therapeutically effective amount. The amount of a drug that is therapeutically effective for the treatment of a particular disease or condition depends on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The exact dose used in the formulation will also depend on the route of administration and the severity of the disease or disorder and should be determined according to the judgment of the attending physician and the circumstances of each patient. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

  The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of a pharmaceutical composition that can be used in a method of treatment. Optionally, such a container may be provided with a notice in the form prescribed by a government agency that regulates the manufacture, use or sale of pharmaceutical or biological products, which notice is for administration to humans. Reflects government approval for manufacturing, use, or sales. The pack or kit can be labeled with information regarding the mode of administration, the order of drug administration (eg, individually, sequentially or simultaneously), and the like. The pack or kit may also include means to remind the patient to receive therapy. The pack or kit can be a single unit dose of the combination therapy or multiple unit doses. Specifically, the drugs can be separated or mixed in any combination present in a single vial or tablet. Drugs packaged in blister packs or other dispensing means are preferred. For the purposes of the present invention, unit dose is intended to mean a dose that is dependent on the individual pharmacodynamics of each drug and is administered at an FDA approved dose during a standard time course.

Transgenic animals or homologous recombinant animals The present invention also relates to the production of non-human transgenic animals. For example, in one embodiment, a host cell that contains a nucleic acid encoding optineurin (eg, a nucleic acid encoding an optineurin polypeptide in a fertilized oocyte or embryonic stem cell) is used. Such host cells can be used to create non-human transgenic animals in which a foreign nucleotide sequence has been introduced into the genome or homologous recombinant animals in which the endogenous nucleotide sequence has been modified. Alternatively, the present invention also relates to the production of non-human animals in which native optineurin is denatured.

  Such animals study the nucleotide sequence and the function and / or activity of the polypeptide encoded by this nucleic acid, and identify modulators of their activity, to investigate the process of optineurin-related glaucoma And / or useful for evaluation. As used herein, a “transgenic animal” is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more cells in the animal contain a transgene. Or a primate. Other examples of transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, and amphibians. A transgene is a foreign DNA that is integrated into the genome of a cell, from which a transgenic animal develops and the DNA remains in the adult genome of the animal, resulting in one or more of the transgenic animal's Expression of the gene product encoded in the cell type or tissue is indicated. As used herein, a “homologous recombinant animal” refers to an endogenous gene and an exogenous DNA molecule (for example, an endogenous gene introduced into an animal cell, such as an embryonic cell of an animal, prior to animal development). A non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous gene has been denatured by homologous recombination with a mutant.

  Methods for producing transgenic animals, particularly animals such as mice, by embryo manipulation and microinjection are routine in the art, e.g., U.S. Pat.Nos. 4,736,866 and 4,870,009, U.S. Pat. Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986). Methods for constructing homologous recombination vectors and homologous recombination animals are described in Bradley, (1991), Current Opinion in Bio / Technology, 2: 823-829 and International Publications WO 90/11354, WO 91/01140, WO It is further described in 92/0968 and WO 93/04169. Non-human transgenic animal clones described herein are described in Wilmut et al. (1997) Nature 385: 810-813 and International Publication Nos. WO 97/07668 and WO 97/07669. It can also be produced according to the method.

  Identification of the association of the optineurin gene with glaucoma and its known interaction with a group of proteins provides the first opportunity to examine the biochemical pathways involved in the pathogenesis of this group of eye diseases. In addition, identifying this gene as a significant contributor to the development of glaucoma allows screening for this disease in high-risk individuals, such as the elderly, as well as prophylactic and therapeutic treatment of the disease .

  The following examples are provided for the purpose of illustrating the invention and should not be understood as limiting the scope of the invention. All references cited herein are hereby incorporated by reference in their entirety.

Identification of Optineurin and Association with POAG Family-derived Substances A large family (Sarfarazi, M. et al., Am. J. Hum) originally used to map the GLC1E locus to 10p14-p15 to screen for mutations A total of 147 surviving affected subjects from 54 families with adult-onset glaucoma including Genet., 62: 641, (1998)) were used. The majority of these families showed only LTG members (i.e., IOP ≤ 22 mmHg), while other families had mixed clinical signs of LTG and moderately elevated IOP (i.e., 23-26 mmHg) in different members showed that. In addition, 124 predominantly LTG and sporadic subjects were used to screen for one optineurin exon mutation.

Identification of optineurin as GLC1E causative gene As described, sequencing of affected subjects was performed directly with the ABI-377 automated DNA sequencer (Stoilova, D. et al., J. Med. Genet., 35: 989, ( 1998)). Sequencing was performed on published linked family samples (Sarfarazi, M. et al., Am. J. Hum. Genet., 62: 641 (1998)). In the initial screening of four candidate genes: IL2RA (interleukin 2 receptor α), IL15RA (interleukin 15 receptor α), GATA3 (GATA binding protein 3) and NAPOR (neuroblast apoptosis-related RNA binding protein) Although no disease-causing mutations were identified, several silent (third base codon) changes, SNPs and insertion / deletion alterations were identified. Examination of the fifth gene and its sequencing identified a missense mutation ( G AG → A AG; E50K) in exon 4 of optineurin (GenBank accession numbers AF420371 to AF420373; SEQ ID NOs: 1, 3, 5 See also). Sequencing of additional affected siblings and more closely related affected relatives confirmed the presence of the E50K mutation in all of them (Table 1).

  Single strand conformation polymorphism (SSCP) assay of E50K mutation showed complete segregation of this large family (49 members including 15 surviving affected individuals). This mutation was not present in 540 normal control chromosomes. An SSCP screening of 54 families with adult-onset glaucoma identified the same E50K mutation in another six families. This mutation showed segregation of 124 members, including 38 affected, 15 asymptomatic gene carriers, 50 unaffected, and 20 spouses. As a result, it was concluded that E50K is a recurrent mutation. Of the 38 affected subjects with the E50K mutation, 7 (ie 18.4%) IOPs were recorded at 23-26 mmHg and IOP values for the remaining individuals ranged from 11-21 mmHg.

  Two additional mutations (2-base pair AG insertion and R545Q) were identified in two other families with normal IOP (Table 1).

A second mutation in exon 6 (insertion of 2 base pairs “ AG ” after ASP127) was observed in LTG subjects. This mutation shifts the reading frame after the insertion point and translates 22 new amino acids until it is finally stopped at a new immature stop codon. This cleaves this protein at 76% of the normal protein.

A third mutation in exon 16 (C G G → C A G; R545Q) was identified in another unrelated LTG subject. This mutation was not present in over 100 normal chromosomes.

The fourth sequence change in exon 5 (A T G → A A G; M98K) was recorded in 23 (ie, 13.6%) of all 169 proband patients (ie, 45 families and 124) Other sporadic and predominantly LTG subjects were screened only for this exon). Only 3 of these 23 subjects had higher IOP values than normal (ie 23, 26 and 40 mmHg) and the remaining 20 subjects had been previously diagnosed with LTG. M98K changes were also present in 9 of 422 normal control chromosomes (ie 2.1%). However, these nine subjects have not undergone comprehensive glaucoma testing, and therefore one or more of them are likely to develop glaucoma at some point. In any case, the observed difference in frequency between affected (13.6%) and normal controls (2.1%) is very significant (X2 = 30.99; df = 1; P = 2.18 × 10 ^-7 ) and degenerative Since amino acids are also conserved in macaques (see below), M98K certainly represents a factor associated with glaucoma risk.

  Taken together, sequence alterations in the optineurin gene may be responsible for 16.67% to 17.98% (32/178) of adult-onset glaucoma (see Table 1 above). Additional mutations may also be present in family and / or sporadic cases. Genotyping and testing of several flanking DNA and optineurin intragenic markers did not identify haplotypes common to these seven families.

Additional sequence modifications in optineurin
Eight additional sequence modifications were identified (see Table 2). These changes were confirmed by sequencing genomic DNA, BAC clones and cDNA prepared from sequencing human trabecular meshwork (HTM) and lymphocytes. The observed changes were consistent across all our samples, but were found to be different from the FIP-2 sample (accession number AF061034).

Optineurin genome and protein structure Optineurin has been mapped to the GLC1E locus, and its physical location is limited to 10p14. As shown in FIG. 1, this gene contains three non-coding exons in its 4 ′ untranslated region (UTR) and another 13 exons encoding a total of 577 amino acids (aa). FIG. 1 shows the approximate regions that interact with other known proteins, including the putative functional domains, the size of each exon, and the location and type of observed mutation. Splicing was confirmed with the 5′-UTR, which resulted in at least three different isoforms (accession number AF420371-3), none of which denatured the code exons.

  Optineurin is a cellular protein that contains two putative bZIP transcription factor basic motifs, several leucine zipper domains, and a C2H2-type Zn finger (FIG. 1). This acidic protein (pI = 5.15) is rich in both glutamic acid (15.8%) and leucine (11.8%).

Other species of optineurin During this study, the mouse optineurin gene was also cloned. The mouse gene encodes 584aa (67 kDa) and shows 78% identity to human optineurin. The mouse gene is also divided into 13 coding exons, the boundaries of which are completely conserved with human genes. By examining public databases, the complete cDNA sequence of cynomolgus optica optineurin (571aa; 65 kDa) and other rats (Moreland, RJ et al., Nucleic Acids Res., 28: 1986, (2000)), porcine and bovine other A partial sequence was identified. Overall, human optineurin has 78% to 85% identity with its homologues of mouse, rat, pig and cow, and 96% identity with macaques. Interestingly, both E50K and M98K mutations observed in 7 and 23 proband patients, respectively, in this study are conserved in humans and macaques. The evolutionary conservation of M98K further confirms that this mutation is a risk factor for glaucoma. The E50K mutation is further conserved in mice and cattle.

Expression of human optineurin in the eye and not in the eye
PCR amplification from HTM, non-pigmented ciliary epithelium (NPCE), retina, brain, adrenal cortex, liver, fetus, lymphocytes and normal dermal fibroblasts (NHDF) and mutant dermal fibroblasts (E50K-DF) In the prepared sample, expression of optineurin was shown. Northern analysis was performed using a radiolabeled optineurin-specific cDNA probe (approximately 2.0 kb) derived from two human cell lines established from (1) trabecular meshwork and (2) non-pigmented ciliary epithelium. Hybridized with 5 micrograms of poly A + RNA. Northern blotting recorded a major band of approximately 2.0 kb message in both HTM and NPCE cell lines. This was 3-4 times more than the 3.6kb message. These transcripts are generally consistent with previously reported messages in the heart, brain, placenta, liver, skeletal muscle, kidney, and pancreas (Li, Y. et al., Mol. Cell. Biol., 18 : 1601, (1998)).

Western analysis Human, macaque, mouse, rat, pig and bovine cDNA sequence alignments showed a significant degree of protein conservation across these species. In addition, selected anti-peptide antibodies were prepared. Chickens were immunized using two different 18 amino acid peptides from the N-terminus (MSHQPLSCLTEKEDSPSE, SEQ ID NO: 7) and C-terminus (EVLPDIDTLQIHVMDCII, SEQ ID NO: 8) of optineurin to obtain anti-optinurin antibodies.

  Standard ELISA, immunoblot, and immunocytochemistry assays were developed using anti-optineurin antibodies. The specificity of the antibody in these assays was recorded by two separate methods. First, non-immune immunoglobulin that did not react in any of the experiments conducted during the study was used as a specificity control. Second, anti-human optineurin antibody was pre-adsorbed using an optineurin specific peptide antibody. Prior adsorption abolished all immunoreactivity in the cells tested.

  These selected anti-peptide antibodies were 100% conserved with known sequences of mouse and macaque optineurin. Western analysis was performed using various cell types. Cells were washed with ice-cold protease inhibitor buffer (1 tablet of Roche protease inhibitor cocktail in 10 ml phosphate buffered saline (PBS)). Cells were lysed by adding protease inhibitor buffer supplemented with 1% CHAPS. Cell lysate (approximately 40 μg protein per lane) was applied to a 4-15% Tris-HCl gradient gel and blotted onto a PVDF membrane. Non-specific hybridization was blocked with 5% dry skim milk in PBST (PBS and 0.5% Tween 20). The membrane was probed at 1/100 with a primary antibody (human optineurin anti-peptide antibody, produced in chicken as described above). After washing, the membrane was incubated at 1: 10,000 with a secondary antibody (rabbit anti-chicken antibody conjugated with HRP) (Sigma). Colorimetric detection was performed using the Opti-4CN kit (Bio-Rad). One of these antibodies cross-reacted with an approximately 66 kDa protein in whole cell extracts from various cell lines including HTM, NPCE, E50K-EF, NHDF and HeLa.

  Since optineurin was detected in both HTM and NPCE, the latter being a component of the secretory epithelium, we decided to determine the expression of optineurin in the aqueous humor. For this purpose, Zoo Western blots were prepared from human and 7 other aqueous humor. Since cloning of the mouse gene predicted a protein size of 67 kDa, the NIH3T3 cell line was used as another subject. All samples showed the presence of proteins of similar size, including those based on the known sequences of human (66 kDa), macaque (65 kDa) and mouse (67 kDa). The presence of this protein was further confirmed with ocular tissue homogenates prepared from selected groups of these animals. These data indicate that optineurin is a secreted protein that is highly conserved during evolution.

Immunocytochemical analysis of Optineurin Both primary cell lines (NHDF and E50K-DF) and transformed cell lines (HTM and NPCE) were used to study the cellular localization of this protein. Immunocytochemical studies have demonstrated particulate staining of the optineurin endogenous protein associated with vesicular structures near the nucleus. Cells were seeded in 6-well plates and grown on glass coverslips for 48 hours (medium was changed once after 24 hours). The cells were then washed twice with PBS, fixed on ice for 20 minutes in 4% paraformaldehyde, washed twice with PBS and permeabilized in 0.1% Triton X-100 for 10 minutes. After washing twice with PBS, non-specific hybridization was blocked with 4% bovine serum albumin in PBS for 30 minutes. Cells were incubated for 1 hour with primary antibody at 1/200. After washing, the cells were incubated for 45 minutes with secondary antibody (goat anti-chicken, Molecular Probes, Inc.) labeled 1/500 with Alexa Fluor 488 (green) or Alexa Fluor 594 (red). For nucleic acid staining, after washing, the cells were incubated with TO-PRO-3 iodine for 30 minutes at 1/200. For staining of the Golgi apparatus, cells were incubated with BODIPY FL C5-ceramide at 1/3000 for 30 minutes. After washing with PBS, cover slips were placed on slides with antifade reagents and examined with a Zeiss 410 laser scanning confocal microscope.

  Consistent perinuclear localization of this endogenous protein was seen in both virus-transformed and non-transformed normal cell lines. Specific staining of the Golgi showed that perinuclear localization of this protein extends to the Golgi complex and vesicle structure. As a control, the use of nonimmune immunoglobulins and peptide antigens specific for optineurin was used. They did not react with any cell type.

  During the study, cultures of normal fibroblasts and E50K mutant fibroblasts grew naturally and equally, but the amount of endogenous protein in the E50K mutant cells was significantly lower than normal cells. Mutant cells were either completely negative for optineurin or very weakly positive. Furthermore, only 10-20% of E40K cells were positive for the optineurin polypeptide compared to 70-80% of normal cells. Furthermore, in very limited E50K positive cells, optineurin polypeptide is less around the nucleus and appears to be less organized. Thus, the effect of the E50K optineurin mutation is thought to reduce synthesis and redistribute protein products in affected cells. In certain other cells examined, optineurin was not well detected in the cytoplasm. Combined with the predicted instability of this protein and its heterogeneous subcellular distribution, optineurin is probably 3 'when it is rapidly secreted or removed from mature cells. The degradation signal in the UTR suggests that it is transiently expressed. This prediction is supported by the fact that the concentration of this protein has accumulated in the cell culture medium over time at levels much higher than those observed in the cells.

Discussion Three degenerations that cause disease in nine adult-onset low-tension glaucoma (LTG) / POAG families have been identified in the optineurin gene, and 23 (first) LTG patients have risk-related degenerations (See Table 1 above). Conservative estimates indicate that mutations in this gene are responsible for 16.67% to 17.98% of all glaucoma patients studied (see Table 1). Since there are up to 1.2 million LTG subjects and up to 2.47 million POAG subjects in the United States alone, screening for optineurin mutations detects more than 200,000 LTG cases and up to 440,000 POAG cases. Individuals up to twice this number may already have this condition without identifiable clinical signs or symptoms. A recurrent mutation (E50K) conserved in mice, cows and macaques was identified in the basic region of the first putative bZIP transcription factor domain. Since the bZIP domain has a basic region for sequence-specific DNA binding, E40K is thought to suppress this potential DNA binding ability of optineurin. A second mutation was found that was an insertion of “AG” in Exon 6 (after Asp127) that shifted the reading frame and cleaved the normal protein at 75%. This cleaved protein is predicted to have lost its normal interaction with optineurin RAB8, TFIIIA, huntingtin and E3-14.7K proteins (see FIG. 1). A third mutation (R545Q) was identified in exon 16. This mutation is not part of a known protein domain, but is close to the only C2H2 Zn finger motif in the optineurin molecule. Since such domains are usually found within transcription factors, the observed mutations are likely to interfere with this potential function of optineurin. Another denaturation (M98K) was observed in exon 5 of optineurin. This degeneration is mainly present in 13.61% of LTG patients and 2.13% of normal controls (p <0.00001). This sequence change is located in the basic domain of the second putative bZIP transcription factor (see Fig. 1) and is also conserved in macaques, so it is considered to be another risk factor for this condition.

To date, optineurin is not known to have significant homology to any known protein. However, its interaction with several other proteins has been established. FIG. 2 provides a diagram illustrating the interaction of optineurin with other proteins and its potential involvement in alternative pathways. The potential involvement of optineurin in two alternative pathways, FAS ligand (left) and TNF-α (right) has been shown. Interaction is indicated by solid arrows and downstream effects are indicated by hollow arrows. An arrow with a circular tip indicates the blocking effect of one protein on another. It has been previously reported that adenovirus E3-14.7K interacts with the last 172 amino acids of optineurin (Li, Y. et al., Mol. Cell. Biol., 18: 1601, (1998)). . This specific interaction can block the protective effect of E3-14.7K against TNF-α cell death induced by its receptors (ie TNFR1 and RIP). TNF-α can also directly induce optineurin expression in a time-dependent manner (id.). This suggests that optineurin is a component of the TNF-α signaling pathway that can shift the balance towards induction of apoptosis. Furthermore, TNF-α has been reported to significantly increase the severity of damage in the optic nerve head of POAG / LTS subjects (Yuan, L. and Neufeld, AH, Glia, 32:42, (2000); Tezel, G. and Wax, MB, J. Neurosci., 20: 8694, (2000)). Production of this cytokine by reactive optic nerve head astrocytes and glial cells can induce excess nitric oxide to be neurotoxic to the axons of retinal ganglion cells (id.). Thus, normal endogenous optineurin suppresses TNF-α production, either directly or through its interaction with other proteins, possibly through a feedback mechanism, resulting in a neuroprotective role for this group of optic neuropathies It is thought that can be fulfilled. Thus, the variant form of optineurin in glaucoma patients is thought to provide insufficient neuroprotection over decades of normal life, resulting in a delayed presentation of this optic neuropathy.

  As shown in FIG. 2, TNF-α involves activating cytoplasmic phospholipase A2 (cPLA2) to release arachidonic acid (AA) and its potential product inflammatory media (Wold, WS, J. Cell Biochem., 53: 329 (1993)). Since E3-14.7K can block this inflammatory response (id.), It is thought that the interaction of optineurin with this protein can also reverse its blocking ability. Thus, involvement of optineurin in the TNF-α pathway can potentially lead to either apoptosis or inflammation. In the third alternative, cytochrome P450 can metabolize AA into bioactive molecules that may be directly related to the glaucoma phenotype. Mutations in cytochrome P4501B1 have previously been shown and supported by primary congenital glaucoma (Stoilov, I. et al., Hum. Mol. Genet., 6: 641, (1997); Stoilov, I. et al., Am. J. Hum. Genet., 62: 573, (1998)). One of the AA metabolites directly related to vasoconstriction and ion transport (McGiff, JC et al., Curr. Opin. Nephrol. Hypertens., 10: 231, (2001)) is 20-hydroxyeicosatetraeno Inic acid (20-HETE). Recurrent vasospasm has been frequently reported in patients with LTG (Gasser, P. et al., Angiology, 41: 214, (1990); Rankin, SJ, Surv. Ophthalmol., 43, Suppl 1: S176, (1999) In addition, vasoconstriction results in decreased production of aqueous humor (Van Buskirk, EM et al., Am. J. Ophthalmol., 109: 511, (1990)), so mutation of optineurin by the AA-P450 pathway is It appears to play a role in the structural damage reported in LTG patients (Caprioli, J. and Spaeth, GL, Am. J. Ophthaltiiol., 97: 730, (1984)). Effectiveness of calcium channel blockers in the treatment of LTG (Netland, PA et al., Am. J. Ophthalmol., 115: 608, (1993)), and expression of optineurin polypeptide observed in human coronary artery cell cultures Further support this hypothesis. Vasospasm is present not only in LTG but also in Raynaud's disease and migraine (Gasser, P. et al., Angiology, 41: 214, (1990)), and these two conditions are frequently reported in high-pressure POAG.

  Interaction of E3-14.7K with caspase-8 (CASP8) can efficiently block Fas ligand-induced apoptosis (Chen, P. et al., J. Biol. Chem., 273: 5815 (1998)) optineurin forms a protein complex with E3-14.7K and CASP8 to inhibit apoptosis, or as previously reported with TNF-α (Li, Y. et al., Mol. Cell. Biol., 18: 1601 (1998)), it is believed that this interaction can reverse the protective effect of E3-14.7K and consequently induce apoptosis. Thus, the interaction of optineurin with E3-14.7K may regulate signaling pathways downstream of both TNF receptors and Fas.

  In addition to E3-14.7K, the C-terminal part of optineurin also interacts with huntingtin, a defective protein of Huntington's disease (HD) (Faber, PW et al., Hum. Mol. Genet., 7: 1463, (1998 )). Huntingtin has been reported to have an anti-apoptotic effect (Wellington, C. L. et al., J. Neural. Transm., Suppl. 1, (2000)). Since both huntingtin and E3-14.7K bind to the C-terminus of optineurin, binding of huntingtin to optineurin may neutralize apoptosis signals normally mediated by optineurin (Li Y. et al., Mol. Cell. Biol., 18: 1601, (1998)). Similarly, E3-14.7K interacts with CASP8 to inhibit FAS ligand-induced apoptosis (Chen, P. et al., J. Biol. Chem., 273: 5815, (1998)). Because cell death induced by expanded polyglutamine repeats in HD requires CASP8 (Sanches, I. et al., Neuron, 22: 623, (1999)), optineurin-huntingtin-CASP8-E3-14.7 The formation of a potential multidimensional protein complex between K may play a common role in neurodegeneration in both HD and POAG. In addition, optineurin indirectly links huntingtin to RAB8, a small GTP hydrolyzing protein that binds to the N-terminal region of optineurin (Hattula, K. and Peranen, J., Curr. Biol., 10 : 1603, (2000)). Since actin and microtubule reorganization by RAB8 directs dramatic changes in cell shape, the complex molecule formed by the RAB8-optineurin-huntingtin interaction is cell morphogenesis, membrane transport (by RAB8) Or likely to play a central role in the control of vesicle transport (by huntingtin). Immunocytochemical localization in the Golgi apparatus of optineurin suggests that protein transport is one of the functions of this molecule. Recently, in a new Xenopus transgenic model, a variant of the RAB8 protein causes retinal degeneration (Moritz, O. et al., Mol. Biol. Cell, 12: 2341, (2001)) It has been shown to be involved in post-Golgi membrane docking in the body (id.).

  The central leucine-rich domain of optineurin (FIG. 1) interacts with the N-terminal part of TFIIIA (Moreland, R. J. et al., Nucleic Acids Res., 28: 1986, (2000)). The latter binds to the internal control sequence of 5S ribosomal DNA and then, in conjunction with TFIIIB and TFIIIC, forms a stable pre-initiation complex for gene transcription by RNA polymerase III. The interaction of optineurin with TFIIIA converts this molecule from an inactive state to an active state, and thus is likely to activate its transcription.

  Optineurin has also been cloned as a NEMO (NF-κB essential modulator or FIP3) -related protein (NRP) but has been shown to have no effect on NF-κB signaling (Schwambom, K. et al., J. Biol. Chem., 275: 22780, (2000)). Phorbol ester induced phosphorylation of optineurin, but at the same time reduced its half-life (id.). This phosphorylation has been reported not to affect the intracellular localization of endogenous optineurin (id.). Although the specific kinase activity responsible for this phosphorylation has not been identified, we have found that optineurin functions within the assembly and activity of two unknown kinases with molecular weights of 85 kDA and 180 kDA. I showed that I can do it.

  The teachings of the references cited herein are incorporated in their entirety.

  While the invention has been illustrated with reference to preferred embodiments, workers skilled in the art will recognize that the invention can be practiced without departing from the spirit and scope of the invention as defined in the appended claims. It will be understood that various changes in form and detail may be made.

FIG. 5 shows the genomic structure of optineurin, including approximate regions that interact with other known proteins, putative functional domains, exon sizes, and the location and type of observed mutations. FIG. 2 shows the interaction of optineurin with other proteins and the potential involvement of optineurin in the alternative pathway of FAS ligand (left) and TNF-α (right). The interaction is indicated by a solid arrow, the downstream effect is indicated by a hollow arrow, and the blocking effect of one protein against another protein is indicated by an arrow with a circle at the tip.

Claims (35)

  SEQ ID NO: 1, oligonucleotide of about 10 to about 50 nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, oligonucleotide of about 10 to about 50 nucleotides of SEQ ID NO: 3, SEQ ID NO: 5, about 10 of SEQ ID NO: 5 An isolated nucleic acid molecule comprising an oligonucleotide of from about 50 nucleotides or a complement of one of the aforementioned nucleic acid molecules, wherein said nucleic acid molecule has a denaturation of at least one nucleotide, said denaturation An isolated nucleic acid molecule that is an indicator that there is an optineurin-related glaucoma or a risk associated with optineurin in glaucoma.   2. The isolated nucleic acid molecule of claim 1, wherein the denaturation results in a change in the sequence of a polypeptide encoded by the nucleic acid molecule.   The modification is from GAG to AAG at codon 50, insertion of AG after codon 127, change from CGG to CAG at codon 545, change from ATG to AAG at codon 98, one complement of the aforementioned modifications 3. An isolated nucleic acid molecule according to claim 2, which is a combination comprising a genetic change or one or more of the aforementioned modifications.   4. The isolated nucleic acid according to any one of claims 1 to 3, wherein the glaucoma is primary open-angle glaucoma.   A purified polypeptide comprising SEQ ID NO: 2, an active fragment of SEQ ID NO: 2, SEQ ID NO: 4, an active fragment of SEQ ID NO: 4, SEQ ID NO: 6, or an active fragment of SEQ ID NO: 6, wherein the polypeptide is at least 1 Is an indicator that there is a risk associated with optineurin-related glaucoma or glaucoma optineurin, wherein the active fragment exhibits at least one function of the optineurin polypeptide. The polypeptide to perform.   The modification is a glutamic acid to lysine modification at codon 50, an immature stop after codon 127, an arginine to glutamine modification at codon 545, a methionine to lysine modification at codon 98, or one of the aforementioned modifications or 6. The purified polypeptide of claim 5, which is a combination comprising a plurality.   The purified polypeptide according to claim 5 or 6, wherein the glaucoma is primary open-angle glaucoma. Degeneration in the optineurin nucleic acid or in the optineurin polypeptide is an indication that the risk associated with optineurin-related glaucoma or glaucoma optineurin is present or absent,
There is a risk associated with optineurin-related glaucoma or glaucoma optineurin in a sample from an individual, including assessing the sample for denaturation in an optineurin nucleic acid, or denaturation in an optineurin polypeptide, or How to detect that it doesn't exist.   9. The method of claim 8, wherein the optineurin nucleic acid comprises a fragment of at least about 10 to about 50 nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.   The modification is a change from GAG to AAG at codon 50, insertion of AG after codon 127, change from CGG to CAG at codon 545, change from ATG to AAG at codon 98, or one of the aforementioned modifications 10. The method according to claim 9, which is a combination comprising a plurality.   11. The method of claim 9 or 10, wherein the evaluation comprises sequencing all or a portion of the optineurin nucleic acid or hybridizing a nucleic acid probe to the optineurin nucleic acid.   9. The method of claim 8, wherein the optineurin polypeptide comprises at least an active fragment of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.   The modification is a glutamic acid to lysine modification at codon 50, an immature stop after codon 127, an arginine to glutamine modification at codon 545, a methionine to lysine modification at codon 98, or one of the aforementioned modifications or 13. The method of claim 12, wherein the method is a combination of multiple.   9. The method of claim 8, comprising detecting denaturation in the optineurin nucleic acid associated with the presence of optineurin-related glaucoma.   The modification is a GAG to AAG change at codon 50, an AG insertion after codon 127, a CGG to CAG change at codon 545, or a combination comprising one or more of the aforementioned modifications. 14. The method according to 14.   16. The method of claim 14 or 15, further comprising diagnosing optineurin-related glaucoma in the individual.   9. The method of claim 8, comprising detecting degeneration in the optineurin nucleic acid associated with the presence of a risk associated with glaucoma optineurin.   18. The method of claim 17, wherein the denaturation is a change from ATG to AAG at codon 98.   19. The method of claim 17 or 18, further comprising diagnosing a glaucoma optineurin-related risk in the individual.   9. The method of claim 8, comprising detecting the absence of denaturation in the optineurin nucleic acid associated with the absence of optineurin-related glaucoma.   9. The method of claim 8, comprising detecting the absence of degeneration in the optineurin nucleic acid associated with the absence of the risk associated with glaucoma optineurin.   The method according to any one of claims 8 to 21, wherein the glaucoma is primary open-angle glaucoma.   A method of treating glaucoma in an individual comprising administering to the individual a therapeutically effective amount of an optineurin therapeutic agent.   A method of treating an individual having an increased risk of glaucoma comprising administering to the individual a therapeutically effective amount of an optineurin therapeutic agent.   Optineurin Therapeutic Agents Alter Optineurin Polypeptide Expression, Alter Optineurin Polypeptide Composition, Alter Optineurin Polypeptide Activity, Alter Postneutrans Polypeptide Processing 25. The agent of claim 23 or 24, selected from the group consisting of an agent, an agent that modulates transcription of an optineurin splicing variant, an optineurin interacting polypeptide, and an agent that alters the expression or activity of an optineurin interacting polypeptide. The method described.   Increased risk associated with optineurin-related glaucoma or glaucoma optineurin in an individual, including detecting the presence or absence of degeneration in expression, composition or activity of an optineurin nucleic acid or optineurin polypeptide The presence of degeneration in expression, composition, or activity is an indicator of increased risk associated with optineurin-related glaucoma or glaucoma optineurin. A method wherein the absence of degeneration in expression, composition, or activity is an indication that there is no increase in risk associated with optineurin-related glaucoma or glaucoma optineurin.   27. The method of claim 26, wherein the optineurin nucleic acid comprises SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, and the optineurin polypeptide comprises SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.   27. The method of claim 26, wherein the modification is a qualitative modification in the composition or activity of an optineurin polypeptide, a quantitative modification in the expression or activity of an optineurin polypeptide, or a combination thereof.   27. The method of claim 26, wherein an antibody that specifically binds to optineurin is used to detect the presence or absence of denaturation in the optineurin polypeptide.   A method of detecting glaucoma or the risk of glaucoma in an individual comprising assessing a sample from the individual for denaturation in an optineurin nucleic acid, denaturation in an optineurin polypeptide, or both.   32. The method of claim 30, wherein the optineurin nucleic acid comprises a fragment of at least about 10 to about 50 nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5.   The modification is a change from GAG to AAG at codon 50, insertion of AG after codon 127, change from CGG to CAG at codon 545, change from ATG to AAG at codon 98, or one of the aforementioned modifications 32. The method of claim 31, wherein the method comprises a combination comprising a plurality.   33. The method of claim 31 or 32, wherein the evaluation comprises sequencing all or a portion of the optineurin nucleic acid, or hybridizing a nucleic acid probe to the optineurin nucleic acid.   32. The method of claim 30, wherein the optineurin polypeptide comprises at least an active fragment of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6. The modification is glutamic acid to lysine modification at codon 50, immature stop after codon 127, arginine to glutamine modification at codon 545, methionine to lysine modification at codon 98, or one of the aforementioned modifications or 35. The method of claim 34, which is a combination comprising a plurality.

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